Application of CHO cell strain overexpressing Bag6 in preparation of medicine for improving expression quantity of exogenous recombinant protein

Abstract

The invention discloses an application of a CHO cell strain overexpressing Bag6 in improving the expression quantity of exogenous recombinant protein, and belongs to the technical field of bioengineering. Based on the fact that Bag6 protein is overexpressed in the cell, the innovation point is that the obtained modified cell has a remarkable enhancement effect on the expression of exogenous recombinant protein; the CHO cell strain for overexpressing the Bag6 in the application technology of the CHO cell strain for overexpressing the Bag6 in improving the expression quantity of the exogenous recombinant protein is characterized in that CHO cells are infected by lentivirus to enable the CHO cells to overexpress the Bag6 gene to construct a stable transfection cell line for overexpressing the Bag6, and the stable transfection cell line for overexpressing the Bag6 is used for expressing the exogenous recombinant protein (an antibody, hemagglutinin, enzyme, green fluorescent protein and the like). And the yield can be greatly increased by 1-10 times.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to an application of a CHO cell line overexpressing Bag6 in the preparation of an exogenous recombinant protein with enhanced expression. Background Art

[0002] Chinese hamster ovary (CHO) cells are the predominant eukaryotic cell expression system for modern commercial production of recombinant proteins. This family of cells originated in the laboratory of Dr. Theodore T. Puck in 1957. Since CHO cells were first used in bioreactors to produce complex large-molecule biopharmaceuticals in 1971, a variety of CHO cell-derived biologics have been approved for marketing, including monoclonal antibodies, interferon, and erythropoietin. However, compared to bacterial or yeast-based expression systems, the production of biopharmaceuticals using mammalian cell expression systems remains subject to certain limitations, such as limited resistance to environmental stress, low productivity, and high culture reagent costs.

[0003] With the continuous emergence of blockbuster biopharmaceuticals, the demand for biopharmaceutical APIs has surged from grams to kilograms and even tons, requiring a simultaneous increase in target protein unit production. By leveraging methods such as large-scale cell (and culture medium) screening based on expression levels, vector design based on optimized target gene expression enhancers or promoters, and metabolic pathway-based cell engineering, researchers have increased the expression level of monoclonal antibody drug engineering cell lines from approximately one gram per liter in the 1990s to an average of three to five grams per liter in recent years, with some even reaching over ten grams per liter.

[0004] Furthermore, technologies such as safe harbor regulation based on site-specific integration, regulation of apoptosis, key factors in the cell cycle and metabolism, and factors involved in protein secretion pathways have also been used in cell engineering research and have achieved considerable progress. The application of these new technologies and methods will pave the way for rationalized CHO cell line development and advanced biomanufacturing of complex proteins.

[0005] With the development of the emerging biopharmaceutical industry, typical monoclonal antibodies are being replaced by novel drug molecules. An increasing number of complex novel protein-based drugs, such as viral antigens, recombinant trimeric subunit vaccines, and bispecific antibodies, are entering the industrial production pipeline. In many cases, these biomolecules are challenging to express, requiring tailored solutions to improve transient or stable expression yields.

[0006] Current approaches are more focused on developing highly integrated, high-throughput screening platforms to screen out more suitable cell clones, better culture media and feeds, and optimal cell culture conditions.

[0007] The CHO genome was sequenced by Xu et al. in 2011. After more than a decade of development, CHO gene annotation and other 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 bioprocessing, thereby identifying new target molecules and revealing their potential mechanisms of action. These targets can be identified through tools such as gain-of-function and loss-of-function techniques, laying the foundation for the development of superior cell-based production plants for difficult-to-express complex proteins.

[0008] However, few researchers have used safe harbor sites to improve the expression yield of recombinant proteins. Summary of the Invention

[0009] In response to the above technical problems, the present invention integrates the results of multi-omics analysis to discover potential safe harbor sites and study their effects on the expression yields of various recombinant proteins. The purpose of the present invention is to provide a modified cell line based on overexpression of Bag6 protein in cells, which has a significant enhancing effect on the expression of exogenous recombinant proteins. The application of the Bag6-overexpressing CHO cell line in the preparation of a technology for increasing the expression of exogenous recombinant proteins is to construct a stable cell line overexpressing Bag6 by infecting CHO cells with a lentivirus to overexpress the Bag6 gene. The stable cell line overexpressing Bag6 is used to express exogenous recombinant proteins (antibodies, hemagglutinins, enzymes, green fluorescent proteins, etc.), which can significantly increase the yield (1-10 times).

[0010] The present invention is achieved through the following technical solutions:

[0011] A use of a CHO cell line overexpressing Bag6 in the preparation of an exogenous recombinant protein with increased expression, wherein the CHO cell line overexpressing Bag6 is prepared by cloning the amino acid sequence of each Chinese hamster Bag6 protein subtype into a eukaryotic expression vector to obtain a recombinant plasmid pRRL-Bag6-Hygro;

[0012] Then, the four plasmids were transferred into 293FT cells using the calcium phosphate transfection method using the lentiviral four-plasmid packaging system. After culturing for 48 hours, the culture supernatant containing Bag6-Hygro lentivirus was harvested.

[0013] The CHO cell line overexpressing Bag6 is a CHO-K1-Bag6 cell overexpressing Bag6; the exogenous recombinant protein is any one of an antibody, a hemagglutinin, an enzyme, and a green fluorescent protein;

[0014] The expression level is increased by 1 to 10 times.

[0015] Preferably, the four-plasmid lentiviral packaging system is pLP1, pLP2, pLP-VSVG, and pRRL-Bag6-Hygro-transfer vector.

[0016] Preferably, the Bag6 protein subtypes of the Chinese hamster are X1 to X13, which are 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 any one of recombinant coagulase rPCE and nuclease SF.

[0020] Preferably, the method for constructing the CHO cell line overexpressing Bag6 is:

[0021] CHO-K1 cells were infected with Bag6-Hyrgo lentivirus and selected with 125 μg / ml hygromycin for 7 days;

[0022] or,

[0023] The exogenous gene was first transferred into CHO-K1 cells, and CHO-K1 cells stably expressing the exogenous gene were obtained after pressure screening. The CHO-K1 cells were then 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 and lentiviral transfection.

[0025] Compared with the prior art, the present invention has at least the following technical effects:

[0026] The present invention provides an application of a CHO cell line overexpressing Bag6 in the preparation of an exogenous recombinant protein with increased expression. The CHO cell line overexpressing Bag6 is constructed by infecting CHO cells with a lentivirus to overexpress the Bag6 gene, and the stably transfected cell line overexpressing Bag6 is constructed. The exogenous recombinant protein (antibody, hemagglutinin, enzyme, green fluorescent protein, etc.) is expressed by the stably transfected cells overexpressing Bag6, which can greatly increase the yield (1-10 times). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is an analysis diagram of the amino acid sequence alignment of each Bag6 isoform;

[0028] Figure 2 Schematic diagram of the construction of plasmid pRRL-Bag6-Hygro;

[0029] Figure 3 Schematic diagram of the yield of green fluorescent protein expressed in CHO-K1 cell line;

[0030] Figure 4 Schematic diagram of increasing the production of green fluorescent protein in the CHO cell line CHO-K1-Bag6 overexpressing Bag6;

[0031] Figure 5 A cell growth curve diagram for increasing the production of antibody Ab1 in the CHO-K1-Bag6 cell line;

[0032] Figure 6 Schematic diagram of antibody expression in the culture supernatant for increasing the production of antibody Ab1 in CHO-K1-Bag6 cell line;

[0033] Figure 7 A cell growth curve diagram for increasing the production of hemagglutinin HA(7-4) in CHO-K1-Bag6 cell line;

[0034] Figure 8 To improve 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 Western blotting;

[0035] Figure 9 A cell growth curve diagram for improving the production of recombinant coagulase rPCE in CHO-K1-Bag6 cell line;

[0036] Figure 10 To improve the production of recombinant coagulase rPCE in CHO-K1-Bag6 cell line, the expression level of recombinant coagulase rPCE in the culture supernatant was detected by Western blotting;

[0037] Figure 11Cell growth curve for improving the production of nuclease SF in CHO-K1-Bag6 cell line;

[0038] Figure 12 In order to improve the production of nuclease SF in CHO-K1-Bag6 cell line, the expression level of nuclease SF in the culture supernatant was detected by protein immunoblotting. DETAILED DESCRIPTION

[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 used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially.

[0040] Example 1:

[0041] Comparison and analysis of amino acid sequences of various Bag6 isoforms, such as Figure 1 Shown is the comparison analysis of the amino acid sequences of each Bag6 subtype:

[0042] There are 13 isoforms (X1-X13) of Bag6 protein in Chinese hamster (Cricetulus griseus). The numbers of X1 to X13 in genbank are: 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 was constructed using the pRRL-EGFP plasmid as its backbone. The pRRL-EGFP plasmid was purchased from our laboratory and detailed in the literature (doi:10.1186 / 1742-4690-7-79.). The Bag6 gene was inserted between XbaI and BamHI, and the Hygromycin gene was inserted between XhoI and ClaI of the pRRL-EGFP plasmid.

[0045] like Figure 2This is the map construction of plasmid pRRL-Bag6-Hygro.

[0046] Plasmid pRRL-Bag6-Hygro was used as a transfer vector for lentiviral packaging to construct Bag6-overexpressing stably transfected cells.

[0047] Example 3: Construction of CHO cell line CHO-K1-Bag6 overexpressing Bag6

[0048] A lentiviral four-plasmid packaging system (pLP1, pLP2, pLP-VSVG, pRRL-Bag6-Hygro) was used. The four plasmids were transferred into 293FT cells using the calcium phosphate transfection method. After 48 hours of culture, the culture supernatant of the lentiviral vector 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 CHO-K1-Bag6 cells overexpressing Bag6 were obtained after 7 days of selection with hygromycin (125 μg / ml).

[0051] Specific methods:

[0052] (1) Take 1×10 CHO-K1 cells 6 Transfer the cells to a 6-well plate, add 3 ml of culture supernatant containing Bag6-Hygro lentivirus, supplement with 5 ml of fresh CD-CHO culture medium, and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0053] (2) Collecting CHO-K1 cells: Place the culture supernatant in a 15 ml centrifuge tube. Add 0.5 ml of 0.25% trypsin to the adherent CHO cells and digest them for 2 minutes. Terminate the digestion reaction with 3 ml of DMEM complete medium and transfer the cells to a 15 ml centrifuge tube.

[0054] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, and resuspend the cells in 5 ml of PBS. Repeat this process once.

[0055] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer the cells to a new 6-well plate, add hygromycin (final concentration of 125 μg / ml), and place in a 37°C, 5% CO2 incubator for 24 hours.

[0056] After 7 days of hygromycin selection, the CHO-K1-Bag6 cell line overexpressing Bag6 was 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, and pRRL-EGFP). CHO-K1 and CHO-K1-Bag6 cells were infected, respectively, and the expression of EGFP was detected by flow cytometry 24 hours later.

[0059] The specific method is as follows:

[0060] (1) Take 1×10 CHO-K1 and CHO-K1-Bag6 cells respectively 5 Transfer the cells to a 6-well plate, add 0.8 ml of culture supernatant containing EGFP lentivirus, supplement with 4 ml of fresh CD-CHO culture medium, and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0061] (2) Collect CHO-K1 and CHO-K1-Bag6 cells: Place the culture supernatant in a 15 ml centrifuge tube. Add 0.5 ml of 0.25% trypsin to the adherent CHO cells and digest them for 2 minutes. Terminate the digestion reaction with 3 ml of DMEM complete medium and transfer the cells to a 15 ml centrifuge tube.

[0062] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently beat the cells, and resuspend the cells in 5 ml of PBS.

[0063] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently beat the cells, and resuspend the cells in 0.5 ml PBS.

[0064] (5) Use flow cytometry to detect the expression of EGFP in CHO-K1 and CHO-K1-Bag6 cells.

[0065] like Figure 3 As shown, the schematic diagram of the production of green fluorescent protein expressed by CHO-K1 cell line; Figure 3 A is a flow cytometric 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 cytometric scatter plot of CHO-K1 cells transformed with the EGFP gene; D is an EGFP fluorescence intensity plot of CHO-K1 cells transformed with the GFP gene; E is the overlap of B and D.

[0066] like Figure 4 As shown, it is a schematic diagram of increasing the production of green fluorescent protein in the CHO cell line CHO-K1-Bag6 overexpressing Bag6, wherein: Figure 4A is a flow cytometric 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 cytometric scatter plot of CHO-K1-bag6 cells transformed with the EGFP gene; D is an EGFP fluorescence intensity plot of CHO-K1-bag6 cells transformed with the EGFP gene; E is the overlap of Figures B and D.

[0067] Combining the results Figure 3 and Figure 4 It was found that compared with the parent CHO-K1 cells, the intensity of EGFP fluorescence in the CHO-K1-Bag6 cell line was greatly improved, and the fluorescence intensity was positively correlated with the concentration of EGFP protein. Therefore, the production of exogenous EGFP protein expressed by CHO-K1-Bag6 was greatly improved.

[0068] Experimental Example 5: CHO-K1-Bag6 cell line can increase the production of antibody Ab1

[0069] Similar to the above, a transfer vector carrying the monoclonal antibody Ab1 gene was used to package and prepare a lentiviral vector containing the Ab1 gene. CHO-K1 and CHO-K1-Bag6 cells were infected for 24 hours, and then blasticidin was used for selection for 7 days to obtain a cell line stably expressing the antibody Ab1. Subsequently, a 14-day Fed-Batch was performed to compare the differences in antibody production.

[0070] The specific method is as follows:

[0071] (1) Take 1×10 CHO-K1 and CHO-K1-Bag6 cells respectively 6 Transfer the cells to a 6-well plate, add 3 ml of the culture supernatant containing Ab1 lentivirus, supplement with 5 ml of fresh CD-CHO culture medium, and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0072] (2) Collect CHO-K1 and CHO-K1-Bag6 cells: Place the culture supernatant in a 15 ml centrifuge tube. Add 0.5 ml of 0.25% trypsin to the adherent CHO cells and digest them for 2 minutes. Terminate the digestion reaction with 3 ml of DMEM complete medium and transfer the cells to a 15 ml centrifuge tube.

[0073] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, and resuspend the cells in 5 ml of PBS. Repeat this process once.

[0074] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer the cells to a new 6-well plate, add blasticidin (final concentration of 7.5 μg / ml), and place in a 37°C, 5% CO2 incubator for 24 hours.

[0075] After 7 days of blasticidin selection, a cell line stably expressing antibody Ab1 was obtained. The cells were transferred to a 125 ml shake flask and cultured at 37° C., 6% CO 2 , and shaking at 90 rpm.

[0076] 14-day Fed-Batch: 0.5×10 6 125 ml shake flask was inoculated with 20 ml AltairCHO medium. On days 3, 5, 7, 9, and 11, 800 μl AltairCHO-Feed and 80 μl CDSF36 were added to maintain the glucose concentration at no less than 3 g / L. The culture supernatant was harvested on day 14.

[0077] BLI detection of the content of antibody Ab1 in the culture supernatant: using a proA sensor and PBST buffer (PBS+0.02% Tween 20) to analyze the antibody content in the supernatant to be tested, with a binding time of 120 s.

[0078] like Figure 5 Shown is a cell growth curve diagram of CHO-K1-Bag6 cell line in increasing the production of antibody Ab1;

[0079] like Figure 6 Shown is a schematic diagram of the antibody expression level in the culture supernatant of the CHO-K1-Bag6 cell line to increase the production of antibody Ab1.

[0080] Combining the results Figure 5 and 6 It was found that the CHO-K1-Bag6 cell line could increase the production of Ab1 antibody by at least 50% compared with the parent CHO-K1 cell line.

[0081] Experimental Example 6: CHO-K1-Bag6 cell line can increase the production of hemagglutinin HA (7-4)

[0082] Similar to the above, a transfer vector carrying the avian influenza hemagglutinin gene HA (7-4) was used to package and prepare a lentiviral vector containing the HA (7-4) gene. After 24 hours, blasticidin was used for selection. After 7 days, a cell line stably expressing hemagglutinin HA was obtained. 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 1×10 CHO-K1 and CHO-K1-Bag6 cells respectively 6 The cells were plated in 6-well plates, 3 ml of culture supernatant containing HA (7-4) lentivirus was added, 5 ml of fresh CD-CHO culture medium was added, and the plates were placed in a 37° C., 5% CO 2 incubator for 24 hours.

[0085] (2) Collect CHO-K1 and CHO-K1-Bag6 cells: Place the culture supernatant in a 15 ml centrifuge tube. Add 0.5 ml of 0.25% trypsin to the adherent CHO cells and digest them for 2 minutes. Terminate the digestion reaction with 3 ml of DMEM complete medium and transfer the cells to a 15 ml centrifuge tube.

[0086] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, and resuspend the cells in 5 ml of PBS. Repeat this process once.

[0087] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer the cells to a new 6-well plate, add blasticidin (final concentration of 7.5 μg / ml), and place in a 37°C, 5% CO2 incubator for 24 hours.

[0088] (5) After 7 days of blasticidin selection, a cell line stably expressing hemagglutinin HA was obtained. The cells were transferred to a 125 ml shake flask and cultured at 37°C, 6% CO2, and 90 rpm shaking.

[0089] (6) 14-day Fed-Batch: 0.5×10 6 Cells / ml, 20 ml Altair CHO medium was inoculated into a 125 ml shake flask, and 800 μl Altair CHO-Feed and 80 μl CDSF36 were supplemented on days 3 / 5 / 7 / 9 / 11 to maintain the glucose concentration at not less than 3 g / L. 100 μl of the culture supernatant was collected on days 3, 4, 5, and 6 and centrifuged at 4000 rpm for 3 minutes.

[0090] Western blotting was used to detect the expression level of hemagglutinin HA (7-4) in the culture supernatant.

[0091] like Figure 7 Shown is a cell growth curve diagram 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 Western blotting in order to increase the production of hemagglutinin HA (7-4) in CHO-K1-Bag6 cell line.

[0093] Combining the results Figure 7 and 8 It was found that the CHO-K1-Bag6 cell line could increase the expression of HA7-4 protein by at least 5-10 times compared with the parental CHO-K1 cells.

[0094] Experimental Example 7: CHO-K1-Bag6 cell line can increase the production of recombinant coagulase rPCE

[0095] Similar to the above, a lentiviral vector containing the recombinant procoagulant (rPCE) gene was packaged and prepared using a transfer vector carrying the rPCE gene. CHO-K1 and CHO-K1-Bag6 cells were infected with the rPCE lentivirus for 24 hours, and then blasticidin was used for selection for 7 days to obtain cell lines stably expressing rPCE. Subsequently, a 14-day Fed-Batch was performed to compare the differences in antibody production.

[0096] The specific method is as follows:

[0097] (1) Take 1×10 CHO-K1 and CHO-K1-Bag6 cells respectively 6 Transfer the cells to a 6-well plate, add 3 ml of the culture supernatant containing rPCE lentivirus, supplement with 5 ml of fresh CD-CHO culture medium, and place in a 37°C, 5% CO2 incubator for 24 hours.

[0098] (2) Collect CHO-K1 and CHO-K1-Bag6 cells: Place the culture supernatant in a 15 ml centrifuge tube. Add 0.5 ml of 0.25% trypsin to the adherent CHO cells and digest them for 2 minutes. Terminate the digestion reaction with 3 ml of DMEM complete medium and transfer the cells to a 15 ml centrifuge tube.

[0099] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, and resuspend the cells in 5 ml of PBS. Repeat this process once.

[0100] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer the cells to a new 6-well plate, add blasticidin (final concentration of 7.5 μg / ml), and place in a 37°C, 5% CO2 incubator for 24 hours.

[0101] (5) After 7 days of blasticidin selection, a cell line stably expressing recombinant coagulase rPCE was obtained. The cells were transferred to a 125 ml shake flask and cultured at 37°C, 6% CO2, and shaking at 90 rpm.

[0102] (6) 14-day Fed-Batch: Inoculate 20 ml of Altair CHO medium into a 125 ml shake flask at 0.5 × 106 cells / ml. Supplement with 800 μl of Altair CHO-Feed and 80 μl of CDSF36 on days 3, 5, 7, 9, and 11 to maintain a glucose concentration of no less than 3 g / L. Collect 100 μl of the culture supernatant on days 3, 4, 5, and 6 and centrifuge at 4000 rpm 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 Shown is a cell growth curve diagram 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 Western blotting in the CHO-K1-Bag6 cell line to improve the production of recombinant coagulase rPCE.

[0106] Combining the results Figure 9 and 10 It was found that compared with the parental CHO-K1 cells, the CHO-K1-Bag6 cell line can significantly increase the expression of rPCE by 5-10 times.

[0107] Experimental Example 8: CHO-K1-Bag6 cell line can increase the production of nuclease SF

[0108] Similar to the above, a transfer vector carrying a recombinant nonspecific nuclease (SF) gene was used to package and prepare a lentiviral vector containing the SF gene. After 24 hours of infection of CHO-K1 and CHO-K1-Bag6 cells with SF lentivirus, blasticidin was used for selection for 7 days to obtain a cell line stably expressing SF. Subsequently, a 14-day Fed-Batch was performed to compare the difference in the production of nuclease SF secreted by the two cell lines.

[0109] The specific method is as follows:

[0110] (1) Take 1×10 CHO-K1-SF cells 6 Transfer the cells to a 6-well plate, add 3 ml of culture supernatant containing Bag6-Hygro lentivirus, supplement with 5 ml of fresh CD-CHO culture medium, and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0111] (2) Collecting CHO-K1-SF cells: Place the culture supernatant in a 15 ml centrifuge tube. Add 0.5 ml of 0.25% trypsin to the adherent CHO cells and digest them for 2 minutes. Terminate the digestion reaction with 3 ml of DMEM complete medium and transfer the cells to a 15 ml centrifuge tube.

[0112] (3) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, and resuspend the cells in 5 ml of PBS. Repeat this process once.

[0113] (4) Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, gently flick the cells, resuspend the cells in 4 ml of CD-CHO medium, transfer the cells to a new 6-well plate, add hygromycin (final concentration of 125 μg / ml), and place in a 37°C, 5% CO2 incubator for 24 hours.

[0114] (5) After 7 days of hygromycin selection, a CHO-K1-SF-Bag6 cell line overexpressing Bag6 was obtained. The cells were transferred to a 125 ml shake flask and cultured at 37°C, 6% CO2, and shaking at 90 rpm.

[0115] (6) 14-day Fed-Batch: 0.5×10 6 Cells / ml, 20 ml Altair CHO medium was inoculated into a 125 ml shake flask. On days 3 / 5 / 7 / 9 / 11, 800 μl Altair CHO-Feed and 80 μl CDSF36 were supplemented to maintain the glucose concentration at not less than 3 g / L. On days 1, 2, 3, 4, and 5, 100 μl of the culture supernatant was collected and centrifuged at 4000 rpm for 3 minutes.

[0116] (7) Western blotting was used to detect the expression level of nuclease SF in the culture supernatant of CHO-K1-SF and CHO-K1-SF-Bag6.

[0117] like Figure 11 Shown is a cell growth curve of CHO-K1-Bag6 cell line in increasing the production of nuclease SF;

[0118] like Figure 12 As shown, the expression level of nuclease SF in the culture supernatant was detected by Western blotting in the CHO-K1-Bag6 cell line to improve the production of nuclease SF.

[0119] Combining the results Figure 11 and 12 It was found that the CHO-K1-Bag6 cell line could significantly increase SF nuclease by 1-5 times compared with the parental CHO-K1 cells.

[0120] Finally, it should be noted that the above description is only 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 shall be included in the scope of protection of the present invention.

Claims

1. A use of a CHO cell line overexpressing Bag6 in the preparation of an exogenous recombinant protein with increased expression, characterized in that: The CHO cell line overexpressing Bag6 is prepared by cloning the amino acid sequence of each Chinese hamster Bag6 protein subtype into a eukaryotic expression vector to obtain the recombinant plasmid pRRL-Bag6-Hygro; Then, the four plasmids were transferred into 293FT cells using the calcium phosphate transfection method using the lentiviral four-plasmid packaging system. After culturing for 48 hours, the culture supernatant containing Bag6-Hygro lentivirus was harvested. The CHO cell line overexpressing Bag6 is a CHO-K1-Bag6 cell overexpressing Bag6; The exogenous recombinant protein is any one of an antibody, hemagglutinin, enzyme, and green fluorescent protein; The expression level is increased by 1 to 10 times.

2. The use according to claim 1, characterized in that The four-plasmid lentiviral packaging system is pLP1, pLP2, pLP-VSVG, and pRRL-Bag6-Hygro-transfer vector.

3. The use according to claim 1, characterized in that The Bag6 protein subtypes of the Chinese hamster are X1 to X13, which are 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.

4. The use according to claim 1, characterized in that The antibody is antibody Ab1.

5. The use according to claim 1, characterized in that The hemagglutinin is hemagglutinin HA (7-4).

6. The use according to claim 1, characterized in that The enzyme is any one of recombinant coagulase rPCE and nuclease SF.

7. The use according to claim 1, characterized in that The method for constructing the CHO cell line overexpressing Bag6 is as follows: CHO-K1 cells were infected with Bag6-Hyrgo lentivirus and selected with 125 μg / ml hygromycin for 7 days; or, The exogenous gene was first transferred into CHO-K1 cells, and CHO-K1 cells stably expressing the exogenous gene were obtained after pressure screening. The CHO-K1 cells were then infected with Bag6-Hyrgo lentivirus and selected with 125 μg / ml hygromycin for 7 days.

8. The use according to claim 7, characterized in that The method for transferring the exogenous gene into CHO-K1 cells includes any one of liposome transfection, electroporation, and lentivirus transfection.

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