Double-gene screening expression vector of CHO monoclonal cell strain as well as preparation method and application of double-gene screening expression vector

Abstract

The invention discloses a double-gene screening expression vector of a CHO monoclonal cell strain and a preparation method and application of the double-gene screening expression vector, the double-gene screening expression vector comprises a glutamyl synthetase GS screening gene and an antibiotic screening gene, the antibiotic screening gene is subjected to weakening expression treatment, that is, an IRES sequence exists in front of the antibiotic screening gene, and an IRES sequence exists in front of the antibiotic screening gene. A target expression gene and an antibiotic screening marker gene share one set of promoter and terminator, and when the double-gene screening expression vector disclosed by the invention is used for screening CHO monoclonal cell strains, the screening efficiency can be remarkably improved, the screening time is saved, and the target gene has high expression quantity.

Description

Technical Field

[0001] The present invention relates to a double-gene screening expression vector for a CHO monoclonal cell line and a preparation method and application thereof, belonging to the field of cell engineering. Background Art

[0002] CHO cells were isolated from adult female hamster ovaries by Dr. Theodore T. Puck of the University of Colorado in 1957. They are epithelial adherent cells. They are immortal and can be passaged for more than a hundred generations. They are currently widely used in bioengineering. Compared with other expression systems, they have the following advantages: (1) They have accurate post-transcriptional modification functions, and the proteins expressed are closest to natural proteins in terms of molecular structure, physicochemical properties, and biological functions; (2) They can grow both adherently and in suspension culture, and can withstand high shear forces and osmotic pressures; (3) They have the ability to efficiently amplify and express recombinant genes, and the integration of exogenous proteins is stable; (4) They have the ability to secrete products extracellularly and rarely secrete their own endogenous proteins, which facilitates the separation and purification of downstream products; (5) They can be cultured at high densities in suspension culture or in serum-free medium, and the culture volume can reach more than 1,000 L, making them suitable for large-scale production.

[0003] There are many CHO cell types, such as DG44, DXB11, CHO K1, and CHO-S. Beginning in the 1980s and 1990s, the DHFR (dihydrofolate reductase-deficient) gene amplification and screening system, using the DG44 host cell line, was an early industry practice. When methotrexate (MTX) is present in the cell culture medium, DHFR is inhibited, leading to feedback regulation that amplifies the gene. Genes within 100-1,000 kb upstream and downstream are also amplified, allowing the target gene to be amplified by inserting it into this site. Many current monoclonal antibody production systems still utilize the DG44 DHFR system. The GS (glutamine synthetase) amplification system, using CHO-K1 as the host cell, is a recently developed, novel gene amplification and screening system that offers significant advantages over the DHFR system and is currently widely recognized and used internationally. Its principle is that GS, while providing energy through ATP hydrolysis, utilizes intracellular ammonia and glutamate to synthesize glutamine. Adding the GS inhibitor L-methioninesulfoximine (MSX) to a culture medium lacking exogenous glutamine can effectively amplify the GS gene and the target gene linked to it, thereby achieving the goal of increasing the expression level of the target gene. The advantages of this system are: (1) it does not require a gene-deficient CHO-K1 cell line as a host cell; (2) CHO-K1 cells are easy to culture and are stronger; (3) it does not require the addition of glutamine to the culture medium, which can avoid the problem of high ammonia levels in the culture system caused by glutamine decomposition, reduce the difficulty of process control, and effectively increase cell fermentation density and prolong cell survival time.

[0004] The pEE series of vectors is a Glutamine Synthetase (GS) gene expression system developed by Lonza Biopharmaceuticals. Utilizing the GS selection marker, they enable high-level expression of target genes in mammalian cells. The most commonly used expression vectors are pEE6.4 and pEE12.4. In our laboratory, we inserted target genes (e.g., GFP subcloned sequences) into pEE12.4 vectors and transfected them into CHO-K1 cells. However, during monoclonal cell line screening, we observed a high false-positive rate, resulting in a screening efficiency of only 40%-50%.

[0005] The Lipofectamine series of reagents are liposome transfection reagents developed by Thermo Fisher Scientific. These reagents demonstrate excellent efficiency, viability, and reproducibility across a wide range of cell types and have been cited over 50,000 times. The most commonly used reagents include Lipofectamine 2000, Lipofectamine 3000, and Lipofectamine LTX. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the screening efficiency is low and the false positive rate is high when using the screening marker of the pEE12.4 vector (ie, GS screening) in screening CHO monoclonal cell lines.

[0007] The present invention provides a method for screening a CHO monoclonal cell line, which comprises the following steps: 1) inserting an antibiotic selection marker gene into a pEE12.4 vector to obtain a modified pEE12.4 multiple cloning site; 2) inserting a target gene into the multiple cloning site of the pEE12.4 modified in step 1) to obtain a constructed recombinant plasmid; 3) transfecting the recombinant plasmid constructed in step 2) into CHO cells using a transfection reagent; 4) performing pressure screening to obtain positive CHO cells; and 5) screening the positive CHO cells to obtain a CHO monoclonal cell line that highly expresses the target protein.

[0008] In the technical solution of the present invention, the antibiotic screening marker gene is a puromycin acetyltransferase gene, a hygromycin phosphotransferase gene, a bleomycin resistance gene, and an aminoglycoside phosphotransferase gene; the preferred antibiotic screening marker gene is the puromycin acetyltransferase gene.

[0009] In the technical solution of the present invention, an IRES sequence is preferably provided between the multiple cloning site of pEE12.4 and the antibiotic selection marker gene.

[0010] In the technical solution of the present invention, the preferred CHO cells are CHO-K1 cells.

[0011] In the technical solution of the present invention, the transfection reagent used when the recombinant plasmid is transfected into CHO cells is lipofectamine 2000, lipofectamine 3000, or Lipofectamine LTX; preferably, Lipofectamine LTX; and preferably, the mass ratio of Lipofectamine LTX to the recombinant plasmid is 9:2.5.

[0012] In the technical solution of the present invention, preferably, 5 μg / ml puromycin and 25 μM MSX are added to the culture medium during the screening of monoclonal cell lines.

[0013] The present invention also provides a method for constructing a eukaryotic expression vector and its application. In a preferred embodiment of the present invention, a GFP gene or a classical swine fever E2 gene is inserted into the modified pEE12.4-IRES-puro plasmid of the present invention to obtain a pEE12.4-GFP-IRES-puro recombinant plasmid and a pEE12.4-E2-IRES-puro recombinant plasmid. This construction process is simple and easily repeatable, and eliminates the need to start from a pEE12.4 plasmid and gradually construct the pEE12.4-GFP-IRES-puro recombinant plasmid or the pEE12.4-E2-IRES-puro recombinant plasmid. Therefore, it can significantly improve work efficiency and reduce costs when constructing eukaryotic expression vectors in batches.

[0014] The method of the present invention can significantly improve the screening efficiency and save screening time when screening CHO monoclonal cell lines. The screening efficiency can be as high as 70%-80%, which is much higher than the 40%-50% when using only the GS screening system. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Plasmid map of pEE12.4-IRES-puro.

[0016] Figure 2 Electrophoresis of IRES-puro PCR results. M: DL5,000; 1: IRES-puro (E1), approximately 1,219 bp.

[0017] Figure 3 Electrophoresis of pEE12.4-IRES-puro plasmid enzyme digestion verification results. M: DL10,000; 1-5: pEE12.4-IRES-puro-1-5 (EcoR I digested, vector size approximately 8788 bp), digested correctly; 6-10: pEE12.4-IRES-puro-12, 14, 18, 20, 22 (EcoR I digested, vector size approximately 8788 bp), digested correctly; 11: Negative control with EcoR I digestion alone.

[0018] Figure 4 Fluorescence detection results of pEE12.4-GFP-IRES-puro recombinant plasmid transfected using three transfection reagents. 4a shows the transfection results using lipofectamine 2000; 4b shows the transfection results using lipofectamine 3000; and 4c shows the transfection results using Lipofectamine LTX. DETAILED DESCRIPTION

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0020] The strains, plasmids, and reagents used in the examples of the present invention are all commercially available products.

[0021] The eukaryotic expression vector PEE12.4 was purchased from Shanghai Linyuan Biotechnology Co., Ltd.

[0022] Puromycin acetyltransferase was kindly donated by Professor Zhou Demin of the State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center.

[0023] The hygromycin phosphotransferase gene (GenBank: X03615) was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the resulting engineered bacteria were labeled DH5α-pUC57-hpt. The bleomycin resistance gene was derived from pcDNA3.1 / Zeo(+) (Invtrogen), and the aminoglycoside phosphotransferase gene was derived from pcDNA3.1 (Invtrogen).

[0024] The IRES sequence was kindly donated by Professor Zhou Demin from the State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center.

[0025] CHO-K1 cells were obtained from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences and the Cell Bank of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences.

[0026] Lipofectamine 2000, lipofectamine 3000, and Lipofectamine LTX were purchased from ThermoFisher.

[0027] The specific implementation method of the present invention is described below using the puromycin acetyltransferase gene (PURO) as an example.

[0028] Example 1: PCR amplification of target fragment IRES-PURO

[0029] 1.1 PCR reaction

[0030] (1) Primer design and synthesis

[0031] Upstream primer: 5'-GGGCACGTGCGGACCGAATTGAATTCAATTCCGCCCCTCTCC-3'

[0032] Downstream primer: 5'-CTGATTATGATCAATGAATTTCAGGCACCGGGCTTGCGGGTCATGCAC-3'

[0033] (2) The sample loading system is 25 μL, as shown in the following table:

[0034]

[0035] PCR amplification procedure:

[0036]

[0037] 1.2 Agarose gel electrophoresis of PCR products

[0038] (1) Prepare agarose gel of a certain concentration according to the size of the PCR product fragment.

[0039] 0.8% agarose gel: Weigh 0.8 g of agarose into a 250 mL blue reagent bottle, add 100 mL of 1× TAE solution, and heat in a microwave for about 2 minutes to dissolve it.

[0040] (2) Prepare the rubber plate, insert the small hole sample comb, and adjust the rubber plate to keep it horizontal.

[0041] (3) After the agarose gel solution has cooled to 40-50°C, add 10 μL of 10 mg / mL ethidium bromide solution to the agarose gel solution and mix well.

[0042] (4) Pour the agarose gel solution into the gel plate. After the agarose gel solidifies, place the agarose gel into the electrophoresis tank and gently pull out the sample comb.

[0043] (5) Take 5 μL of PCR product and mix it with 0.5 μL of 10× loading buffer and add it to the agarose gel sample well. Take 5 μL of DNA molecular weight standard reagent as a standard for analyzing the size of DNA fragments.

[0044] (6) Confirm that the electrode direction is correct, cover the electrophoresis tank, adjust the electrophoresis instrument voltage to 95V, constant voltage, electrophoresis for 15-20 minutes, and turn off the electrophoresis instrument.

[0045] (7) The imager displays the image, analyzes the results and saves the image. Figure 2 shown.

[0046] 1.3 Gel recovery of PCR products

[0047] (1) Label the sample collection EP tube, adsorption column, and collection tube.

[0048] (2) Weigh the marked empty EP tube and record the value.

[0049] (3) Carefully cut the single target DNA band from the agarose gel using a scalpel on a gel cutter and place it into a clean 1.5 mL centrifuge tube.

[0050] (4) Add 600 μL of PC buffer to the 1.5 mL centrifuge tube in step (3) and place in a 50°C water bath for about 5 minutes. During this time, gently flip the centrifuge tube upside down to ensure that the gel is fully dissolved.

[0051] (5) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CB2 (the adsorption column is pre-placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0052] (6) Add the solution obtained in step (5) to the adsorption column CB2, let it stand for 2 minutes, centrifuge at 10,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and then place the adsorption column CB2 into the collection tube.

[0053] (7) Add 600 μL of PW buffer to the adsorption column, let it stand for 3 min, centrifuge at 10,000 rpm for 30 s, discard the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube.

[0054] (8) Repeat step (7).

[0055] (9) Centrifuge the empty adsorption column at 12,000 rpm for 2 min to remove as much rinse solution as possible. Leave the adsorption column at room temperature for 10 min to dry thoroughly.

[0056] (10) Place the adsorption column CB2 in the collection tube, add 50 μL of Lution Buffer (preheated at 65°C) to the middle of the adsorption membrane, let it stand for 3 minutes, and centrifuge at 12,000 rpm for 2 minutes.

[0057] (11) Remove the centrifuge tube from step (10) from the centrifuge, discard the adsorption column CB2 in the middle, cover the centrifuge tube, and retain the DNA sample in the centrifuge tube.

[0058] (12) Store the DNA sample in step 11 at 4°C and prepare agarose gel electrophoresis to identify the DNA fragments recovered from the gel.

[0059] Example 2: Vector single enzyme digestion reaction

[0060] 2.1 Enzyme digestion reaction

[0061] (1) Label the 1.5 mL EP tubes to be used, add the sample and mix thoroughly according to the following table: For a 50 μL reaction system, add the sample as shown in the following table:

[0062]

[0063] (2) Place the 1.5 mL EP tube in step (1) in a 37°C constant temperature water bath for 2-3 hours.

[0064] 2.2 Gel recovery of double enzyme digestion products

[0065] Remove the double enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein, using the same method as 1.3.

[0066] Example 3: Ligation reaction

[0067] (1) Label the 0.2 mL centrifuge tubes to be used.

[0068] (2) Add 20 μL of the reaction system in the following table to a well-labeled 0.2 mL tube:

[0069]

[0070] (3) After adding the sample, gently pipette several times to mix the components.

[0071] (4) Place the 0.2 mL centrifuge tube at 37°C for 30 min. After the reaction is complete, immediately place the tube in an ice water bath to cool for 5 min.

[0072] (5) The reaction product of step (4) can be directly used for transformation experiments, or it can be stored at -20°C and thawed for transformation when needed.

[0073] Example 4: Conversion reaction

[0074] (1) Quickly add 10 μL of ligation reaction solution to 100 μL of competent cells, pipette to mix, and incubate on ice for 30 minutes.

[0075] (2) After step (1) is completed, remove the sample tube, place it in a 42°C water bath for 100 seconds, and then immediately place it in an ice bath for 2 minutes.

[0076] (3) After step (2) is completed, take out the sample tube, add 600 μL of liquid LB culture medium to the sample tube in a clean bench, and then place the sample tube in a 37°C constant temperature shaker at 220 rpm for 1 hour.

[0077] (4) Prepare transformation plates. Prepare LB resistance plates for transformation based on the resistance of the plasmid.

[0078] (5) Plate coating: Take out the sample tube in step (3), centrifuge at 8,000 rpm for 2 min at room temperature, remove 600 μL of supernatant, and resuspend the bacteria at the bottom of the tube with the remaining supernatant. Place the resuspended bacterial solution in the center of the corresponding transformation plate and spread the bacterial solution in the center of the transformation plate evenly with a bacterial spreader.

[0079] (6) Place the plate prepared in step (5) upright in a biochemical constant temperature incubator and incubate at 37°C for 1 hour. Then, invert the transformation plate and incubate for 15 hours.

[0080] (7) Observe and record the transformation results.

[0081] Example 5: Plasmid extraction and double enzyme digestion identification

[0082] 5.1 Plasmid extraction

[0083] (1) Use a 10 μL pipette tip to pick a single colony from the transformation plate and transfer it to 5 ml of LB liquid medium containing ampicillin resistance. Incubate the culture at 37°C and 220 rpm overnight.

[0084] (2) Pipette the bacterial solution into a 1.5 mL EP tube and centrifuge at room temperature, 12,000 rpm, for 2 min. Discard the supernatant.

[0085] (3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) to thoroughly suspend the bacteria.

[0086] (4) Add 250 μL of P2 buffer to the solution from step (3) and immediately mix thoroughly by gently inverting the tube 5-10 times. Let stand at room temperature for 2-4 minutes.

[0087] (5) Add 350 μL of P3 buffer to the solution from step (4) and immediately mix thoroughly by gently inverting the tube 5-10 times. Let stand at room temperature for 2-4 minutes.

[0088] (6) Centrifuge the solution from step (5) at room temperature, 14,000 rpm, for 10 min.

[0089] (7) Transfer the supernatant solution from step (6) to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm, 30 s, and discard the liquid in the collection tube.

[0090] (8) Add 500 μL of Buffer DW1 to the center of the adsorption column and centrifuge at room temperature, 12,000 rpm, for 30 s. Discard the liquid in the collection tube.

[0091] (9) Add 500 μL of wash solution to the center of the column and centrifuge at room temperature, 12,000 rpm, for 30 seconds. Discard the liquid in the collection tube. Repeat once.

[0092] (10) Empty adsorption column, centrifuge at room temperature, 12,000 rpm, 2 min.

[0093] (11) Place the adsorption column in a clean 1.5 ml centrifuge tube, add 30 μL of Elution Buffer to the center of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at room temperature, 12,000 rpm, for 2 min, and store the DNA solution in the tube at 4°C.

[0094] 5.2 Enzyme digestion identification

[0095] (1) Label the 1.5 mL EP tubes to be used, add the sample and mix according to the following table. The reaction system is 20 μL:

[0096]

[0097] (2) Place the 1.5 mL EP tube in step (1) in a 37°C constant temperature water bath for 2 hours.

[0098] (3) The sample in step (2) was subjected to agarose gel electrophoresis to check whether the inserted fragment size was correct. Figure 3 shown.

[0099] (4) Select the clone with the correct insert and send it to a sequencing company for sequencing.

[0100] Example 6: Construction of pEE12.4-GFP-IRES-puro and pEE12.4-E2-IRES-puro

[0101] 6.1 Amplification of target gene fragments

[0102] Using the recombinant plasmid pEGFP-N1 or the synthetic nucleotide sequence of the classical swine fever E2 protein as a template, appropriate primers were designed to amplify the GFP or E2 subclone fragment (containing HindIII and EcoRI restriction sites at both ends, respectively) by PCR. Specific operations are shown in Example 1.

[0103] 6.2 Double enzyme digestion reaction

[0104] The pEE12.4-IRES-puro vector and the GFP fragment or the E2 fragment were double-digested with Hind-III and EcoRI-HF, and then recovered by gel excision. Detailed procedures are described in Example 5.

[0105] 6.3 Ligation reaction

[0106] The GFP fragment or E2 fragment obtained by PCR cloning was ligated with the recombinant plasmid pEE12.4-IRES-puro. Detailed procedures are shown in Example 3.

[0107] Example 7: CHO-K1 cell transfection

[0108] (1) Preparation: Sterilize in a biosafety cabinet with UV for 30 min; preheat DMEM / F12 (containing 10% serum and 1% double antibody), DMEM / F12, and PBS to 37°C in a 37°C water bath.

[0109] (2) Remove cells (10 cm cell culture dish) from the 37°C incubator, discard the supernatant culture medium, wash the cells once with 8 ml of pre-warmed PBS, and discard the PBS.

[0110] (3) Add 1-2 ml of 0.25% trypsin-EDTA to each 10 cm cell culture dish and digest for about 2 minutes at room temperature. Observe under a microscope to see that the cells are wrinkled and rounded and appear as single cells.

[0111] (4) Add 4 ml of DMEM / F12 (containing 10% serum and 1% double antibody) to terminate the digestion reaction, and use a pipette to blow away the cells.

[0112] (5) Transfer the digested cells to a 15 ml centrifuge tube and centrifuge at room temperature, 200 g, for 5 min.

[0113] (6) Resuspend the cells in DMEM / F12 (containing 10% serum and 1% double antibody) and count.

[0114] (7) Dilute cells to 2×10 5 2 ml of the mixed cells were added to a six-well plate, and the six-well plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated overnight.

[0115] (8) Take out the cell culture dish prepared in step (7) and observe the cell status: when the cell confluence reaches 80%-90%, transfection can be started. Before transfection, replace the culture medium with antibiotic-free and serum-free DMEM / F12, 2 mL / well.

[0116] (9) Plasmid dilution: Dilute the plasmid with OPTI-MEM. Add 2.5 μg of pEE12.4-GFP-IRES-puro recombinant plasmid or pEE12.4-E2-IRES-puro to 125 μl of OPTI-MEM, then add 2.5 μl of plus, mix well, and let stand at room temperature for 5 min.

[0117] (10) Dilute lipofectamine 2000, lipofectamine 3000, or Lipofectamine LTX according to the instructions. For example, add 9 μl of Lipofectamine LTX to 125 μl of OPTI-MEM, then add 2.5 μl of plus, mix gently, and let stand at room temperature for 5 min.

[0118] (11) Gently mix the mixture from step (9) and step (10). Let it stand at room temperature for 5 minutes, then add it dropwise to a six-well plate and distribute evenly.

[0119] (12) Place the six-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 4-6 hours.

[0120] (13) Change medium: discard the supernatant medium, add 2ml DMEM / F12 (containing 10% serum and 1% double antibody), and place the six-well plate in a 37℃, 5% CO2 cell culture incubator for overnight. Take pictures with a fluorescence microscope 24 hours after transfection. The results are as follows Figure 4 As shown, Lipofectamine LTX has the highest transfection efficiency.

[0121] Example 8: Pressure Screening (Separately Screening Cells Containing the Two Plasmids Transfected in Example 7)

[0122] 24 hours after transfection with Lipofectamine LTX, pressurization was started: cells in the six-well plate were removed from the 37°C incubator, the supernatant culture medium was discarded, and 2 ml of DMEM / F12 (containing 10% serum + 5 μg / ml puromycin + 25 μM MSX) was added. Pressurization was continued for 7 days, and the cells were observed in the middle. The medium was changed frequently for dead cells.

[0123] Example 9: Monoclonal Screening (Screening Monoclonal Cell Lines Containing the Two Plasmids Transfected in Example 7)

[0124] (1) When negative control cells are almost completely dead, about 7 days later, monoclonal screening begins.

[0125] (2) Remove the six-well plate, discard the culture medium, wash once with PBS, then add 300 μl of 0.25% trypsin-EDTA, digest at room temperature for about 2 min, add 2 ml of DMEM / F12 (containing 10% serum + 5 μg / ml puromycin + 25 μM MSX) to terminate the digestion reaction, and blow the cells apart with a pipette.

[0126] (3) Transfer the digested cells to a 15 ml centrifuge tube and centrifuge at room temperature, 200 g, for 5 min.

[0127] (4) Resuspend the cells in DMEM / F12 (containing 10% serum + 5 μg / ml puromycin + 25 μM MSX) and count.

[0128] (5) Plating: Dilute cells to 5 cells / ml, take 200 μL of the mixed cells and add them to a 96-well plate, and place it in a 37°C, 5% CO2 cell culture incubator for 4-6 hours.

[0129] (6) Recording of single cell wells.

[0130] (7) When single cells have grown in the wells of the 96-well plate, discard the culture medium, wash once with PBS, add 100 μl of 0.25% trypsin-EDTA, and digest at room temperature for about 2 minutes. Add 2 ml of DMEM / F12 (containing 10% serum + 5 μg / ml puromycin + 25 μM MSX) to terminate the digestion reaction, and use a pipette to disperse the cells. Transfer the cell solution to a 12-well plate. When the 12-well plate is full, remove the supernatant and test whether the clone is positive by ELISA. Highly expressing positive clones will continue to be expanded and frozen.

[0131] (8) After calculation, it was found that when using this system to screen monoclonal cell lines, the screening efficiency of monoclonal cell lines expressing two proteins (GFP or E2) can reach 70%-80%.

[0132] (9) After the screened cell lines are acclimated, cultured, and fermented, the protein is purified and the concentration of the purified protein is determined by the BCA method. The total amount of purified protein is calculated based on the total volume of the purified protein, and the protein yield is calculated based on the volume of the cell supernatant used. The purity is detected by HPLC.

[0133] The yield of GFP protein is about 3-5 g / L, and the purity is above 95%.

[0134] The yield of E2 protein is about 0.8-1.1 g / L, and the purity is above 95%.

[0135] Example 10: Insertion of other antibiotic selection marker genes into the pEE12.4 vector and screening of CHO monoclonal cell lines

[0136] 10.1 Recombinant Plasmid Construction

[0137] (1) According to the methods of Examples 1 to 5, other antibiotic selection marker genes, such as hygromycin phosphotransferase gene, bleomycin resistance gene, aminoglycoside phosphotransferase gene, etc., were constructed on the pEE12.4 vector.

[0138] (2) According to the method of Example 6, the target gene is added to the constructed recombinant plasmid, such as the GFP gene and classical swine fever E2 gene added in Example 6.

[0139] 10.2 CHO Monoclonal Cell Line Screening

[0140] (1) According to the methods of Examples 7 to 9, CHO monoclonal cell lines that highly express the target protein were screened. During the screening, only the antibiotic screening reagents added were slightly different. The specific antibiotic screening reagents added are shown in the following table:

[0141]

[0142] (2) After calculation, it was found that the screening efficiency of monoclonal cell lines screened with different antibiotics and marker genes was basically the same, reaching 70%-80%, with little effect on protein yield and purity.

[0143] The present invention is illustrated by the above examples, however, it should be understood that the present invention is not limited to the specific examples and embodiments described herein. These specific examples and embodiments are included herein to assist those skilled in the art in practicing the present invention. Further improvements and modifications will readily occur to those skilled in the art without departing from the spirit and scope of the present invention, and the present invention is therefore limited only by the content and scope of the appended claims, which are intended to cover all alternatives and equivalents included within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A dual-gene screening expression vector for a CHO monoclonal cell line, characterized in that: The dual-gene screening expression vector comprises a glutamine synthetase GS screening gene and an antibiotic screening marker gene, so that the glutamine synthetase GS screening gene forms a multiple cloning site expression vector; an IRES sequence is also present between the multiple cloning site expression vector and the antibiotic screening marker gene for attenuated expression processing.

2. The dual gene screening expression vector according to claim 1, characterized in that: The target expression gene is inserted into the multiple cloning site expression vector, the IRES sequence is between the multiple cloning site expression vector and the antibiotic selection marker gene, and the target expression gene and the antibiotic selection marker gene share a set of promoters and terminators to obtain a recombinant plasmid glutamine synthetase GS selection gene-target expression gene-IRES-antibiotic selection marker gene.

3. The dual gene screening expression vector according to claim 1, characterized in that: The antibiotic screening marker genes are hygromycin phosphotransferase gene, bleomycin resistance gene, aminoglycoside phosphotransferase gene and puromycin acetyltransferase gene.

4. The dual gene screening expression vector according to claim 3, characterized in that: The antibiotic screening marker gene is a puromycin acetyltransferase gene.

5. The dual gene screening expression vector according to claim 1, characterized in that: The expression vector of the glutamine synthetase GS screening gene is a pEE series expression vector.

6. The dual gene screening expression vector according to claim 5, characterized in that: The pEE series expression vectors are pEE6.4 expression vector and pEE12.4 expression vector.

7. The dual gene screening expression vector according to claim 5, characterized in that: The pEE series expression vector is a pEE12.4 expression vector.

8. The dual gene screening expression vector according to claim 6, characterized in that: The target expression genes are GFP gene and classical swine fever E2 gene.

9. A method for preparing the dual-gene screening expression vector according to any one of claims 1 to 8, characterized in that: The preparation method comprises the following steps: 1) Inserting an antibiotic selection marker gene into a pEE series expression vector to obtain a modified pEE multiple cloning site expression vector, wherein an IRES sequence is further provided between the pEE multiple cloning site expression vector and the antibiotic selection marker gene; 2) Insert the target expression gene into the multiple cloning site expression vector to obtain a recombinant plasmid glutamine synthetase GS screening gene-target expression gene -IRES- Antibiotic selection marker genes.

10. The preparation method according to claim 9, characterized in that The pEE series expression vectors are pEE6.4 expression vector and pEE12.4 expression vector; the antibiotic screening marker genes are hygromycin phosphotransferase gene, bleomycin resistance gene, aminoglycoside phosphotransferase gene and puromycin acetyltransferase gene.

11. Use of the dual-gene screening expression vector according to any one of claims 1 to 8 in screening CHO monoclonal cell lines.

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