Application of CHO cell lines overexpressing Bag6 in preparing products with enhanced expression levels of exogenous recombinant proteins
By overexpressing Bag6 protein in CHO cells to construct a stable cell line, and then infecting CHO cells with lentivirus, the problem of low recombinant protein yield in CHO cell systems was solved, and the yield of exogenous recombinant proteins was significantly improved. This method is suitable for expressing antibodies, hemagglutinin, enzymes, and green fluorescent protein.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING INSTITUTE OF PETROCHEMICAL TECHNOLOGY
- Filing Date
- 2025-07-03
- Publication Date
- 2026-06-30
AI Technical Summary
Existing CHO cell systems suffer from low yields, limited ability to withstand environmental stresses, and high costs of culture reagents when producing recombinant proteins, making it difficult to meet the demand for large-scale production of complex and novel proteins.
By overexpressing the Bag6 protein in CHO cells, a stable cell line overexpressing Bag6 was constructed. CHO cells were then infected with lentivirus to overexpress the Bag6 gene, thereby increasing the expression level of the exogenous recombinant protein.
It significantly increases the yield of exogenous recombinant proteins by 1-10 times, and is suitable for expressing antibodies, hemagglutinins, enzymes and green fluorescent proteins, thus solving the shortcomings of CHO cell systems in terms of yield and stability.
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Figure CN120738286B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of a CHO cell line overexpressing Bag6 in the preparation of a method to enhance the expression level of exogenous recombinant proteins. Background Technology
[0002] Chinese hamster ovary (CHO) cells are the most important eukaryotic cell expression system for the modern commercial production of recombinant proteins. This family of cells was first developed in 1957 in the laboratory of Dr. Theodore T. Puck. Since 1971, when CHO cells were used in bioreactors to produce complex macromolecular biopharmaceuticals, various CHO cell-derived biopharmaceuticals have been approved for marketing, including monoclonal antibodies, interferons, and erythropoietin. However, compared to bacterial or yeast-based expression systems, the use of mammalian cell expression systems for biopharmaceutical production still faces certain limitations, such as limited resilience to environmental stress, low productivity, and high costs of culture reagents.
[0003] With the continuous emergence of blockbuster biopharmaceuticals, the demand for biopharmaceutical raw materials has been leaping from the gram level to the kilogram level and even the ton level, which requires a corresponding increase in the unit yield of target proteins. Researchers have used methods such as large-scale cell (and culture medium) screening based on expression levels, vector design based on the optimization of target gene expression enhancers or promoters, and cell engineering based on metabolic pathways to increase the expression level of monoclonal antibody drugs in engineered cell lines from about one gram per liter in the 1990s to an average of three to five grams per liter in recent years, with some even reaching more than ten grams per liter.
[0004] Furthermore, techniques based on site-specific integration technology, such as safe harbor site regulation, apoptosis, key factors in the cell cycle and metabolism, and regulation of factors related to protein secretion pathways, have also been applied to cell engineering research and have achieved some progress. The application of these new technologies and methods will pave the way for the rational development of CHO cell lines and the advanced biomanufacturing of complex proteins.
[0005] With the development of the emerging biopharmaceutical industry, typical monoclonal antibody drugs are gradually being replaced by novel drug molecules. An increasing number of complex novel protein drugs, such as viral antigens, recombinant trimeric subunit vaccines, and bispecific antibodies, are entering industrial production pipelines. In most cases, these biomolecules are difficult to express, requiring tailored solutions to improve their transient or stable expression yields.
[0006] Current approaches focus more on developing highly integrated, high-throughput screening platforms to screen for more suitable cell clones, better culture media and feed, and optimal cell culture conditions.
[0007] The CHO genome sequencing was completed by Xu et al. in 2011, and after more than a decade of development, CHO gene annotation and other related work have become increasingly sophisticated. Recent advances in multi-omics technologies, such as transcriptomics, proteomics, and metabolomics, have enabled the systematic analysis and characterization of important intracellular pathways and processes in biological processing, thereby identifying new target molecules and revealing their potential mechanisms of action. The identification of these target molecules can be achieved through gain-of-function or loss-of-function techniques, laying the foundation for establishing superior cellular production facilities for complex proteins that are difficult to express.
[0008] However, few researchers have conducted research on using safe harbor sites to increase the expression yield of recombinant proteins. Summary of the Invention
[0009] To address the aforementioned technical problems, this invention integrates multi-omics analysis results to identify potential safe harbor sites and investigate their impact on the expression yield of various recombinant proteins. The purpose of this invention is to provide an innovative method based on the significant enhancement of exogenous recombinant protein expression in modified cells obtained by overexpressing Bag6 protein in cells. The application of this Bag6-overexpressing CHO cell line in the preparation of technologies that enhance the expression of exogenous recombinant proteins involves constructing a stable overexpressing Bag6 cell line by infecting CHO cells with lentivirus to overexpress the Bag6 gene. Using these stable overexpressing Bag6 cells to express exogenous recombinant proteins (antibodies, hemagglutinins, enzymes, green fluorescent protein, etc.) can significantly increase the yield (1-10 times).
[0010] This invention is achieved through the following technical solution:
[0011] The application of a CHO cell line overexpressing Bag6 in preparing a recombinant protein with enhanced expression levels is described in this paper. The CHO cell line overexpressing Bag6 is prepared by cloning the amino acid sequences of various Chinese hamster Bag6 protein subtypes into a eukaryotic expression vector to obtain the recombinant plasmid pRRL-Bag6-Hygro.
[0012] Then, using a lentiviral four-plasmid packaging system, the four plasmids were transfected into 293FT cells using the calcium phosphate transfection method. After culturing for 48 hours, the culture supernatant containing Bag6-Hygro lentivirus was harvested.
[0013] The CHO cell line overexpressing Bag6 is CHO-K1-Bag6 cells overexpressing Bag6; the exogenous recombinant protein is any one of antibody, hemagglutinin, enzyme, and green fluorescent protein.
[0014] The expression level increased by 1 to 10 times.
[0015] Preferably, the lentivirus four-plasmid packaging system is pLP1, pLP2, pLP-VSVG, and pRRL-Bag6-Hygro-transfer vector.
[0016] Preferably, the Bag6 protein subtype of the Chinese hamster is X1 to X13, namely XP_035315758.1, XP_027244593.1, XP_027244594.1, XP_027244595.1, XP_027244596.1, XP_027244597.1, XP_035307662.1, XP_027244598.1, XP_035307663.1, XP_027244599.1, XP_027244601.1, XP_027244600.1, and XP_035307665.1.
[0017] Preferably, the antibody is antibody Ab1.
[0018] Preferably, the hemagglutinin is hemagglutinin HA (7-4).
[0019] Preferably, the enzyme is either recombinant coagulase rPCE or nuclease SF.
[0020] Preferably, the method for constructing the CHO cell line overexpressing Bag6 is as follows:
[0021] CHO-K1 cells were infected with Bag6-Hyrgo lentivirus and selected after 7 days of screening with 125 μg / ml hygromycin.
[0022] or,
[0023] First, the exogenous gene was transferred into CHO-K1 cells. After stress selection, CHO-K1 cells that stably expressed the exogenous gene were obtained. Then, CHO-K1 cells were infected with Bag6-Hyrgo lentivirus and selected with 125 μg / ml hygromycin for 7 days.
[0024] Preferably, the method for transferring the exogenous gene into CHO-K1 cells includes any one of liposome transfection, electroporation, or lentivirus transfection.
[0025] Compared with the prior art, the present invention has at least the following technical effects:
[0026] This invention provides an application of a CHO cell line overexpressing Bag6 in preparing and increasing the expression level of exogenous recombinant proteins. This CHO cell line overexpressing Bag6 is constructed by infecting CHO cells with lentivirus to overexpress the Bag6 gene and building a stable cell line overexpressing Bag6. Using stable cells overexpressing Bag6 to express exogenous recombinant proteins (antibodies, hemagglutinin, enzymes, green fluorescent protein, etc.) can significantly increase the yield (1-10 times). Attached Figure Description
[0027] Figure 1 This is a diagram showing the amino acid sequence alignment analysis of each Bag6 subtype;
[0028] Figure 2 This is a schematic diagram illustrating the construction of plasmid pRRL-Bag6-Hygro;
[0029] Figure 3 A schematic diagram illustrating the yield of green fluorescent protein expressed in the CHO-K1 cell line;
[0030] Figure 4 A schematic diagram illustrating the enhancement of green fluorescent protein production in the CHO-K1-Bag6 cell line overexpressing Bag6.
[0031] Figure 5 Cell growth curves for increasing antibody Ab1 production in CHO-K1-Bag6 cell lines;
[0032] Figure 6 A schematic diagram of antibody expression levels in the culture supernatant used to increase the production of antibody Ab1 in CHO-K1-Bag6 cell line.
[0033] Figure 7 Cell growth curves for increasing hemagglutinin HA(7-4) production in CHO-K1-Bag6 cell line;
[0034] Figure 8 To enhance the production of hemagglutinin HA(7-4) in CHO-K1-Bag6 cell line, the expression level of hemagglutinin HA(7-4) in the culture supernatant was detected by protein immunoblotting.
[0035] Figure 9 Cell growth curves for increasing the production of recombinant coagulase rPCE in CHO-K1-Bag6 cell line.
[0036] Figure 10 To increase the yield of recombinant coagulase rPCE in CHO-K1-Bag6 cell line, protein immunoblotting was used to detect the expression level of recombinant coagulase rPCE in the culture supernatant.
[0037] Figure 11Cell growth curves for increasing the yield of nuclease SF in CHO-K1-Bag6 cell line;
[0038] Figure 12 To increase the yield of nuclease SF in CHO-K1-Bag6 cell line, protein immunoblotting was used to detect the expression level of nuclease SF in the culture supernatant. Detailed Implementation
[0039] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0040] Example 1:
[0041] Amino acid sequence alignment analysis of each Bag6 subtype, such as Figure 1 The image shown is a sequence alignment analysis of amino acids from various Bag6 subtypes.
[0042] There are 13 subtypes (X1-X13) of the Bag6 protein in the Chinese hamster (Cricetulus griseus). The genbank numbers for X1 to X13 are as follows: XP_035315758.1, XP_027244593.1, XP_027244594.1, XP_027244595.1, XP_027244596.1, XP_027244597.1, XP_035307662.1, XP_027244598.1, XP_035307663.1, XP_027244599.1, XP_027244601.1, XP_027244600.1, XP_035307665.1.
[0043] Example 2: Design of plasmid pRRL-Bag6-Hygro
[0044] pRRL-Bag6-Hygro uses pRRL-EGFP plasmid as its backbone. pRRL-EGFP plasmid is owned by our laboratory and can be found in the literature (doi:10.1186 / 1742-4690-7-79.). The Bag6 gene is inserted between XbaI and BamHI in the pRRL-EGFP plasmid, and the Hygromycin gene is inserted between XhoI and ClaI, thus completing the construction of the pRRL-Bag6-Hygro plasmid.
[0045] like Figure 2Mapping of plasmid pRRL-Bag6-Hygro.
[0046] The plasmid pRRL-Bag6-Hygro was used as a transfer vector for lentiviral packaging to construct stable cells overexpressing Bag6.
[0047] Example 3: Construction of CHO-K1-Bag6 cell line overexpressing Bag6
[0048] A lentiviral quad plasmid packaging system (pLP1, pLP2, pLP-VSVG, pRRL-Bag6-Hygro) was used. The four plasmids were transfected into 293FT cells using the calcium phosphate transfection method. After 48 hours of culture, the lentiviral vector culture supernatant containing the Bag6-Hygro gene was harvested.
[0049] The specific plasmid nucleotide sequences are shown in SEQ ID NO.1-5.
[0050] CHO-K1 cells were infected with the Bag6-Hyrgo lentiviral vector and selected with hygromycin (125 μg / ml) for 7 days to obtain CHO-K1-Bag6 cells overexpressing Bag6.
[0051] The specific method is as follows:
[0052] (1) Take 1×10 CHO-K1 cells 6 Add 3 ml of culture supernatant containing Bag6-Hygro lentivirus to each 6-well plate, supplement with 5 ml of fresh CD-CHO medium, and incubate at 37°C in a 5% CO2 incubator for 24 hours.
[0053] (2) Collect CHO-K1 cells: Place the culture supernatant in a 15ml centrifuge tube, add 0.5ml of 0.25% trypsin to the adherent CHO cells for 2 minutes, stop the digestion reaction with 3ml of DMEM complete culture medium, and transfer the cells to a 15ml centrifuge tube.
[0054] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently pat the cells apart, resuspend the cells in 5 ml of PBS, and repeat once.
[0055] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently break up the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer to a new 6-well plate, add hygromycin (final concentration of 125 μg / ml), and incubate at 37°C in a 5% CO2 incubator for 24 hours.
[0056] Seven days after hygromycin screening, CHO-K1-Bag6 cell lines overexpressing Bag6 were obtained.
[0057] Example 4: CHO-K1-Bag6 cell line can increase the production of green fluorescent protein.
[0058] Lentiviral vectors carrying the EGFP gene were prepared using a lentiviral four-plasmid packaging system (pLP1, pLP2, pLP-VSVG, pRRL-EGFP) and used to infect CHO-K1 and CHO-K1-Bag6 cells, respectively. The expression level of EGFP was detected by flow cytometry after 24 hours.
[0059] The specific method is as follows:
[0060] (1) Take 1×10⁶ CHO-K1 and CHO-K1-Bag6 cells respectively. 5 Add 0.8 ml of lentivirus culture supernatant containing EGFP to each well of a 6-well plate, supplement with 4 ml of fresh CD-CHO medium, and incubate at 37°C in a 5% CO2 incubator for 24 hours.
[0061] (2) Collect CHO-K1 and CHO-K1-Bag6 cells: Place the culture supernatant in a 15ml centrifuge tube, add 0.5ml of 0.25% trypsin to the adherent CHO cells for 2 minutes, stop the digestion reaction with 3ml of DMEM complete culture medium, and transfer the cells to a 15ml centrifuge tube.
[0062] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently pat the cells apart, and resuspend the cells in 5 ml of PBS.
[0063] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently break up the cells, and resuspend the cells in 0.5 ml of PBS.
[0064] (5) The expression of EGFP in CHO-K1 and CHO-K1-Bag6 cells was detected by flow cytometry.
[0065] like Figure 3 The diagram shows the yield of green fluorescent protein expressed in the CHO-K1 cell line; where, Figure 3 In the diagram, A is a flow cytometry scatter plot of CHO-K1 blank control cells; B is an EGFP fluorescence intensity plot of CHO-K1 blank control cells; C is a flow cytometry scatter plot of CHO-K1 cells transfected with the EGFP gene; D is an EGFP fluorescence intensity plot of CHO-K1 cells transfected with the GFP gene; E is the overlap of plots B and D.
[0066] like Figure 4 The diagram illustrates how overexpressing Bag6 in the CHO cell line CHO-K1-Bag6 increases the production of green fluorescent protein. Figure 4In the diagram, A is a flow cytometry scatter plot of CHO-K1-bag6 blank control cells; B is an EGFP fluorescence intensity plot of CHO-K1-bag6 blank control cells; C is a flow cytometry scatter plot of CHO-K1-bag6 cells transfected with the EGFP gene; D is an EGFP fluorescence intensity plot of CHO-K1-bag6 cells transfected with the EGFP gene; E is the overlap of plots B and D.
[0067] Results combined Figure 3 and Figure 4 It was found that the intensity of EGFP fluorescence in the CHO-K1-Bag6 cell line was significantly increased compared with that of the parent CHO-K1 cells. Since the fluorescence intensity was positively correlated with the concentration of EGFP protein, the production of exogenous EGFP protein in CHO-K1-Bag6 cells was significantly increased.
[0068] Experimental Example 5: CHO-K1-Bag6 cell line can increase the production of antibody Ab1.
[0069] Similar to the above, lentiviral vectors containing the Ab1 gene were prepared by packaging transfer vectors carrying the monoclonal antibody Ab1 gene. After infecting CHO-K1 and CHO-K1-Bag6 cells for 24 hours, cell lines stably expressing the Ab1 antibody were obtained after screening with blastomycin for 7 days. Subsequently, a 14-day Fed-Batch was conducted to compare the differences in antibody yield.
[0070] The specific method is as follows:
[0071] (1) Take 1×10⁶ CHO-K1 and CHO-K1-Bag6 cells respectively. 6 Add 3 ml of culture supernatant containing Ab1 lentivirus to each 6-well plate, supplement with 5 ml of fresh CD-CHO medium, and incubate at 37°C in a 5% CO2 incubator for 24 hours.
[0072] (2) Collect CHO-K1 and CHO-K1-Bag6 cells: Place the culture supernatant in a 15ml centrifuge tube, add 0.5ml of 0.25% trypsin to the adherent CHO cells for 2 minutes, stop the digestion reaction with 3ml of DMEM complete culture medium, and transfer the cells to a 15ml centrifuge tube.
[0073] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently pat the cells apart, resuspend the cells in 5 ml of PBS, and repeat once.
[0074] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently break up the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer to a new 6-well plate, add blast fungicide (final concentration of 7.5 μg / ml), and incubate at 37℃ in a 5% CO2 incubator for 24 hours.
[0075] After 7 days of screening for blast fungicide, a cell line stably expressing antibody Ab1 was obtained. The cells were then transferred to 125 ml shake flasks and cultured at 37°C, 6% CO2, and 90 rpm with shaking.
[0076] 14-day Fed-Batch: 0.5 x 10 6 Inoculate 20 ml of AltairCHO medium into a 125 ml shake flask. On days 3, 5, 7, 9, and 11, supplement with 800 μl of AltairCHO-Feed and 80 μl of CDSF36 to maintain a glucose concentration of at least 3 g / L. Harvest the culture supernatant on day 14.
[0077] BLI assay for antibody Ab1 content in culture supernatant: The antibody content in the supernatant was analyzed using a proA sensor and PBST buffer (PBS + 0.02% Tween 20) with a binding time of 120 s.
[0078] like Figure 5 The figure shows the cell growth curve of the CHO-K1-Bag6 cell line in increasing the production of antibody Ab1.
[0079] like Figure 6 The diagram shows the antibody expression level in the culture supernatant of CHO-K1-Bag6 cell line to enhance antibody Ab1 production.
[0080] Results combined Figure 5 and 6 It was found that, compared with the parental CHO-K1 cells, the CHO-K1-Bag6 cell line could increase the production of Ab1 antibody by at least 50%.
[0081] Experimental Example 6: CHO-K1-Bag6 cell line can increase the production of hemagglutinin HA(7-4).
[0082] Similar to the above, lentiviral vectors containing the HA(7-4) gene were prepared by packaging transfer vectors carrying the avian influenza hemagglutinin gene HA(7-4). After 24 hours, cell lines stably expressing hemagglutinin HA were obtained by screening with blastomycin for 7 days. Subsequently, a 14-day Fed-Batch was performed to compare the differences in antibody production.
[0083] The specific method is as follows:
[0084] (1) Take 1×10⁶ CHO-K1 and CHO-K1-Bag6 cells respectively. 6 Add 3 ml of lentivirus culture supernatant containing HA(7-4) to each well of a 6-well plate, supplement with 5 ml of fresh CD-CHO medium, and incubate at 37°C in a 5% CO2 incubator for 24 hours.
[0085] (2) Collect CHO-K1 and CHO-K1-Bag6 cells: Place the culture supernatant in a 15ml centrifuge tube, add 0.5ml of 0.25% trypsin to the adherent CHO cells for 2 minutes, stop the digestion reaction with 3ml of DMEM complete culture medium, and transfer the cells to a 15ml centrifuge tube.
[0086] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently pat the cells apart, resuspend the cells in 5 ml of PBS, and repeat once.
[0087] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently break up the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer to a new 6-well plate, add blast fungicide (final concentration of 7.5 μg / ml), and incubate at 37℃ in a 5% CO2 incubator for 24 hours.
[0088] (5) After screening for blast fungicide for 7 days, a cell line that stably expresses hemagglutinin HA was obtained. The cells were transferred to a 125ml shake flask and cultured at 37℃, 6% CO2, and 90rpm.
[0089] (6) 14-day Fed-Batch: 0.5 × 10 6 Inoculate 20 ml of AltairCHO medium into a 125 ml shake flask. On days 3 / 5 / 7 / 9 / 11, supplement with 800 μl of AltairCHO-Feed and 80 μl of CDSF36 to maintain a glucose concentration of not less than 3 g / L. On days 3, 4, 5, and 6, collect 100 μl of culture supernatant and centrifuge at 4000 rpm for 3 minutes.
[0090] Protein immunoblotting was used to detect the expression level of hemagglutinin HA(7-4) in the culture supernatant.
[0091] like Figure 7 The figure shows the cell growth curve of CHO-K1-Bag6 cell line in increasing the production of hemagglutinin HA(7-4).
[0092] like Figure 8 As shown, the expression level of hemagglutinin HA(7-4) in the culture supernatant was detected by protein immunoblotting to enhance the production of hemagglutinin HA(7-4) in CHO-K1-Bag6 cell line.
[0093] Results combined Figure 7 and 8 It was found that, compared with the parental CHO-K1 cells, the CHO-K1-Bag6 cell line could increase the expression of HA7-4 protein by at least 5-10 times.
[0094] Experimental Example 7: CHO-K1-Bag6 cell line can increase the production of recombinant coagulase rPCE.
[0095] Similar to the above, lentiviral vectors containing the rPCE gene were prepared by packaging transfer vectors carrying the recombinant procoagulant (rPCE) gene. After infecting CHO-K1 and CHO-K1-Bag6 cells with rPCE lentivirus for 24 hours, cell lines stably expressing rPCE were obtained after 7 days of screening with blastomycin. Subsequently, a 14-day Fed-Batch was conducted to compare the differences in antibody production.
[0096] The specific method is as follows:
[0097] (1) Take 1×10⁶ CHO-K1 and CHO-K1-Bag6 cells respectively. 6 Add 3 ml of lentivirus culture supernatant containing rPCE to each of the 1 to 6 well plates, supplement with 5 ml of fresh CD-CHO medium, and incubate at 37°C in a 5% CO2 incubator for 24 hours.
[0098] (2) Collect CHO-K1 and CHO-K1-Bag6 cells: Place the culture supernatant in a 15ml centrifuge tube, add 0.5ml of 0.25% trypsin to the adherent CHO cells for 2 minutes, stop the digestion reaction with 3ml of DMEM complete culture medium, and transfer the cells to a 15ml centrifuge tube.
[0099] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently pat the cells apart, resuspend the cells in 5 ml of PBS, and repeat once.
[0100] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently break up the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer to a new 6-well plate, add blast fungicide (final concentration of 7.5 μg / ml), and incubate at 37℃ in a 5% CO2 incubator for 24 hours.
[0101] (5) After screening for blast fungicide for 7 days, a cell line that stably expresses recombinant coagulase rPCE was obtained. The cells were transferred to a 125ml shake flask and cultured at 37℃, 6% CO2, and 90rpm.
[0102] (6) 14-day Fed-Batch: Inoculate 0.5×106 cells / ml with 20ml Altair CHO medium into a 125ml shake flask. On days 3 / 5 / 7 / 9 / 11, supplement with 800μl Altair CHO-Feed and 80μl CDSF36 to maintain a glucose concentration of not less than 3g / L. On days 3, 4, 5 and 6, take 100μl of culture supernatant and centrifuge at 4000rpm for 3 minutes.
[0103] (7) Protein immunoblotting was used to detect the expression level of recombinant coagulase rPCE in the culture supernatant.
[0104] like Figure 9 The figure shows the cell growth curve of CHO-K1-Bag6 cell line in increasing the production of recombinant coagulase rPCE.
[0105] like Figure 10 As shown, the expression level of recombinant coagulase rPCE in the culture supernatant was detected by protein immunoblotting to enhance the yield of recombinant coagulase rPCE in CHO-K1-Bag6 cell line.
[0106] Results combined Figure 9 and 10 It was found that, compared with the parental CHO-K1 cells, the CHO-K1-Bag6 cell line could significantly increase rPCE expression by 5-10 times.
[0107] Experimental Example 8: CHO-K1-Bag6 cell line can increase the yield of nuclease SF.
[0108] Similar to the above, lentiviral vectors containing the SF gene were prepared by packaging transfer vectors carrying recombinant nonspecific nuclease (SF) genes. After infecting CHO-K1 and CHO-K1-Bag6 cells with SF lentivirus for 24 hours, cell lines stably expressing SF were obtained after 7 days of screening with blastomycin. Subsequently, a 14-day Fed-Batch comparison was conducted to compare the differences in the production of SF nuclease secreted by the two cell lines.
[0109] The specific method is as follows:
[0110] (1) Take 1×10 CHO-K1-SF cells from each cell. 6 Add 3 ml of culture supernatant containing Bag6-Hygro lentivirus to each 6-well plate, supplement with 5 ml of fresh CD-CHO medium, and incubate at 37°C in a 5% CO2 incubator for 24 hours.
[0111] (2) Collect CHO-K1-SF cells: Place the culture supernatant in a 15ml centrifuge tube, add 0.5ml of 0.25% trypsin to the adherent CHO cells for 2 minutes, stop the digestion reaction with 3ml of DMEM complete medium, and transfer the cells to a 15ml centrifuge tube.
[0112] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently pat the cells apart, resuspend the cells in 5 ml of PBS, and repeat once.
[0113] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently break up the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer to a new 6-well plate, add hygromycin (final concentration of 125 μg / ml), and incubate at 37°C in a 5% CO2 incubator for 24 hours.
[0114] (5) After 7 days of screening with hygromycin, a cell line of CHO-K1-SF-Bag6 overexpressing Bag6 was obtained. The cells were transferred to a 125ml shake flask and cultured at 37℃, 6% CO2, and 90rpm.
[0115] (6) 14-day Fed-Batch: 0.5 × 10 6 Inoculate 20 ml of Altair CHO medium into a 125 ml shake flask. On days 3 / 5 / 7 / 9 / 11, supplement with 800 μl of Altair CHO-Feed and 80 μl of CDSF36 to maintain a glucose concentration of not less than 3 g / L. On days 1, 2, 3, 4 and 5, take 100 μl of culture supernatant and centrifuge at 4000 rpm for 3 minutes.
[0116] (7) Protein immunoblotting detection of the expression level of nuclease SF in the culture supernatant of CHO-K1-SF and CHO-K1-SF-Bag6.
[0117] like Figure 11 The figure shows the cell growth curve of CHO-K1-Bag6 cell line in increasing the yield of nuclease SF.
[0118] like Figure 12 As shown, the expression level of nuclease SF in the culture supernatant was detected by protein immunoblotting to enhance the yield of nuclease SF in CHO-K1-Bag6 cell line.
[0119] Results combined Figure 11 and 12 It was found that, compared with the parental CHO-K1 cells, the CHO-K1-Bag6 cell line can significantly increase SF nuclease by 1-5 times.
[0120] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. The application of a CHO cell line overexpressing Bag6 in enhancing the expression level of exogenous recombinant proteins, characterized in that, The CHO cell line overexpressing Bag6 was obtained by cloning the amino acid sequence of the Bag6 X2 isoform protein from Chinese hamster into a eukaryotic expression vector to obtain the recombinant plasmid pRRL-Bag6-Hygro. The Genebank number for the Bag6 X2 subtype protein is XP_027244593.1; Then, using a lentiviral four-plasmid packaging system, the four plasmids were transfected into 293FT cells using the calcium phosphate transfection method. After culturing for 48 hours, the culture supernatant containing Bag6-Hygro lentivirus was harvested. The method for constructing the CHO cell line overexpressing Bag6 is as follows: CHO-K1 cells were infected with Bag6-Hyrgo lentivirus and selected after 7 days of screening with 125 μg / ml hygromycin. or, First, the exogenous gene was transferred into CHO-K1 cells. After stress selection, CHO-K1 cells that stably expressed the exogenous gene were obtained. Then, CHO-K1 cells were infected with Bag6-Hyrgo lentivirus and selected with 125 μg / ml hygromycin for 7 days. The CHO cell line overexpressing Bag6 is CHO-K1-Bag6 cells overexpressing Bag6 X2 isoform protein; The exogenous recombinant protein is any one of antibody, hemagglutinin, enzyme, and green fluorescent protein; The expression level increased by 1 to 10 times; The lentivirus four-plasmid packaging system consists of pLP1, pLP2, pLP-VSVG, and pRRL-Bag6-Hygro-transfervector; SEQ ID NO:1-3 are helper plasmids pLP1, pLP2, and pLP-VSVG, respectively, and SEQ ID NO:5 is pRRL-Bag6-Hygro-transfer vector plasmid.
2. The application according to claim 1, characterized in that, The enzyme is either recombinant coagulase rPCE or nuclease SF.
3. The application according to claim 1, characterized in that, The method for transferring exogenous genes into CHO-K1 cells includes any one of liposome transfection, electroporation, or lentivirus transfection.