Method for rapidly constructing IgG antibody high-expression stably-transfected CHO cell strain

Abstract

The invention is applicable to the technical field of biotechnology and cell biology, and provides a method for quickly constructing an IgG antibody high-expression stably-transfected CHO cell strain, which comprises the following steps: S1, constructing antibody plasmids; s2, carrying out cell resuscitation, passage and plasmid transfection; s3, screening positive cells; s4, preparing, namely screening high-expression stably transfected single cell clones; s5, cell feeding batch culture; s6, verifying the stability of the cell strain expressed by the antibody, according to the method provided by the invention, after the plasmid vector enters the cell through electrotransfection and standing culture is carried out for 48 hours, shaking culture is carried out, the cell recovery time can be shortened, the recovery speed is higher than that of traditional standing culture, and the cell state is better than that of a standing state; according to the method, high-density cells are inoculated during cell fed-batch culture, and under the high-density inoculation, the logarithmic phase of the cells is shortened, and the cells enter a protein production phase earlier, so that higher expression quantity can be achieved, the overall production period is greatly shortened, and a large amount of cost is saved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and cell biology, and in particular relates to a method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies. Background Art

[0002] Mammalian cells are the primary host cells used to produce commercial therapeutic protein products, including monoclonal antibodies. Chinese hamster ovary (CHO) cells are the preferred host cell for producing protein drugs. Compared with other expression systems, the CHO expression system has significant advantages: (1) Suitable for suspension culture and can meet the requirements of large-scale industrial production of recombinant proteins; (2) It has accurate post-transcriptional modification functions, and the expressed proteins, including antibodies, are closer to natural proteins; (3) Ability to efficiently amplify and express exogenous genes and to fully integrate into cells; (4) CHO cells are fibroblasts that secrete almost no endogenous proteins and have extracellular secretion function, which facilitates the purification of downstream products.

[0003] The construction of a stable cell line refers to the process in which exogenous genes enter the recipient cell and integrate into the cell's chromosomes, enabling long-term and stable expression of the target protein in the host cell. Stable cell lines play a very important role in biological research and are widely used in applications such as recombinant protein and monoclonal antibody production, drug screening, and gene function research.

[0004] Traditional recovery relies on DMSO, which leads to cytotoxicity (viability ≤ 90%). After electroporation, static culture of traditional stably transfected cell lines prolongs recovery time. MSX screening results in a low proportion of positive cells (≤ 65%) due to delayed pressurization and high pressure throughout the entire process. Low-density fed-batch inoculation leads to delayed production cycles. The present method can greatly shorten cell recovery time, replace static culture with shaking culture, and then treat the cells to achieve excellent recovery. At the same time, traditional cells have a low initial density during fed-batch inoculation, while the method provided by the present invention inoculates cells at a high density during cell fed-batch culture. At such a high density, the cells' logarithmic growth phase is shortened, entering the protein production phase earlier, thereby achieving higher expression levels. Summary of the Invention

[0005] The present invention provides a method for rapidly constructing a stable CHO cell line with high expression of IgG antibodies, aiming to solve the above-mentioned problems. The present invention is applicable to the construction of stable cell lines of various IgG subtype antibodies, including but not limited to IgG1, IgG2, and IgG4.

[0006] The present invention is achieved by a method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies, comprising the following steps: S1. Antibody plasmid construction: The antibody light and heavy chains are linked to a vector capable of transfecting CHO (e.g., CHO-K1) cells by whole gene synthesis, and the antibody plasmid is transformed and extracted. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; S2. CHO cell recovery, passaging and plasmid transfection; S3. Positive cell screening: Use MSX pressure screening to obtain positive cells that have successfully transferred the plasmid and establish a cell pool; S4. Preparation and screening of high-expressing stable single-cell clones; S5. Fed-batch cell culture and antibody expression purification: The stably transfected cells with high expression were cultured in a fed-batch format. After the feeding phase, the protein was purified using an AKTA protein purifier to verify the high expression level. S6. Verification of antibody expression cell line stability: High-expressing stable single-cell clones selected by S4 were passaged and frozen; high-expressing stable single-cell clones selected by S4 were passaged to the 20th generation and then frozen. Finally, the first generation and 20th generation frozen cells were revived and cultured in batches through cell feeding, paying attention to the cell status and production stability at this stage.

[0007] Preferably, the S2.CHO cell recovery, passaging and plasmid transfection include the following steps: 1) Take out the CHO cells from the liquid nitrogen tank, thaw them quickly in a 37°C water bath, centrifuge and discard the supernatant, and resuspend the CHO cells in basal medium supplemented with 4 mM glutamine and count them (usually 0.4-0.6×10 6 cells / mL) and then cultured in E125 shake flasks; 2) CHO cells were passaged every other day for a total of 3 passages; 3) The day before transfection, cells were passaged to an appropriate density. The next day, plasmid transfection was performed using a Neon electroporator. The cell number was 4 × 10 6 cells / mL, sample volume was 100 μL, electroporation conditions were: 1500-1700 v / 15-25 ms / 1 pulse, and static culture was performed in a T25 flask with a volume of 5 mL.

[0008] Preferably, the basal medium for S2 cell recovery is supplemented with: 1-2% trehalose, 0.05-0.1 mM sodium selenite and 3-8 mM L-proline.

[0009] Trehalose (cryoprotection): forms a glassy structure and reduces ice crystal damage; Sodium selenite (antioxidant): activates glutathione peroxidase (GPx4) to repair oxidative damage; L-proline (osmotic pressure regulation): stabilizes the conformation of cell membrane proteins and resists the shock of resuscitation osmotic pressure; A ternary composite protective agent is used instead of traditional DMSO, the traditional static step is eliminated, and the cells are directly shaken and cultured after recovery.

[0010] Preferably, the S3. positive cell screening comprises the following steps: after 2 days of static culture in a T25 flask, pressurizing with MSX, transferring from the T25 flask to a 50 mL TPP shake tube for shaking culture, adding about 2 mL of fresh culture medium every other day for the first 5 days, and pressurizing at the same time, and changing the medium by semi-centrifugation every other day for the next 5 days, resuspending in the same volume, and pressurizing with MSX. During the treatment of the cells, the number, viability and cell diameter of the cells are recorded. After about 2 days, the cells can proliferate normally, and positive cells stably transfected with the plasmid are obtained.

[0011] Preferably, a metabolic activator combination is added in the S3 positive cell screening, including: 1-5mM sodium butyrate and 0.5-2mM valproic acid, to enhance the chromatin openness and integration efficiency of the exogenous gene.

[0012] Metabolic activators are added simultaneously in S3 (MSX pressure screening stage) to regulate cellular energy metabolism and chromatin openness, breaking through the traditional method of enriching positive cells solely by screening pressure (MSX). Through metabolic intervention, it actively promotes the chromosomal integration of exogenous genes and activates histone acetylation, which can improve the transcription efficiency of antibody genes, solve the inefficiency problem caused by random integration in the construction of stable transfectants, and shorten the screening cycle.

[0013] Preferably, the S4. preparation and screening of high-expressing stably transfected single cell clones comprises the following steps: 1) Preparation of monoclonal cells: After stable passage, count the cells and plate 1 cell per well in 96-well plates for a total of 10 plates. 2) Screening of monoclonal cells: Place the 96-well plate in a CO2 incubator for static culture. Observe the cells after two hours and mark the wells with single cells. Replenish the culture medium once every 12-14 days. Use ELISA to screen the wells with high OD values ​​and a single monoclonal cell population. 3) Monoclonal expansion: Add fresh culture medium and continue culturing. The next day, transfer the cells in the 96-well plate to a 24-well plate for expansion.

[0014] Preferably, the S5. cell fed batch culture comprises the following steps: inoculating 2.5-3.5×10 6 cells / mL, the total volume can be 15-30 mL, and the yield can be obtained by feeding with feed medium for 12-14 days.

[0015] Specifically, the S5 fed-batch culture can be fed in stages: a) Day 0-4: supplement medium with 6-8 g / L glucose and 2× amino acids; b) Day 5-10: supplement medium with 1-2 g / L glucose and 3% lipid precursor; c) Day 11-14: Supplement with sugar-free medium containing 5 mM sodium butyrate.

[0016] Lipid precursors (such as choline / inositol): enhance cell membrane stability and prolong production period; Sodium butyrate: induces cell cycle arrest in the later stage, reduces fragmentation, and maintains viability for a longer time.

[0017] At such a high density of cell fed-batch culture, the logarithmic growth phase of cells is shortened, and the cells enter the plateau phase of protein production earlier. Through fed-batch culture, a higher viable cell density can be achieved, thereby obtaining a higher protein expression level.

[0018] Compared with the prior art, the embodiments of the present application have the following beneficial effects: 1. The method provided by the present invention for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies is as follows: after the plasmid vector is electroporated into the cells, after 48 hours of static culture, shaking culture is performed, which can accelerate the cell recovery time, recover faster than traditional static culture, and the cell state is also better than the static state.

[0019] 2. The method provided by the present invention for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies is to inoculate cells at a high density during fed-batch culture. Under such a high-density inoculation, the logarithmic growth phase of the cells is shortened, and the cells enter the protein production phase earlier, thereby achieving a higher expression level.

[0020] 3. The method provided by the present invention for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies greatly shortens the overall production cycle and saves a lot of costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of a method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies provided by the present invention; Figure 2 This is a diagram showing the purification results of the strain with the highest expression level obtained by the method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies provided by the present invention using an AKTA purification instrument. DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The terms used in the specification of the application are only for the purpose of describing specific embodiments and are not intended to limit this application. The terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0023] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0024] Example 1 The present invention provides a method for rapidly constructing a stable CHO cell line with high expression of IgG antibodies. Figure 1 As shown, the following steps are included: S1. Antibody plasmid construction: The antibody light and heavy chains are linked to a vector capable of transfecting CHO (e.g., CHO-K1) cells by whole gene synthesis, and the antibody plasmid is transformed and extracted. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; M6 heavy chain variable region amino acid sequence (SEQ ID NO: 1): EVKLVESGGGLVKPGGTLKLSCAASDSSFSTYAMSWIRQTPEKRLEWVASISSGGVTYYQDNVRRGRFTISRDNARNILYLQMSSLRSEDTATYYCARGYFYFDYWGQGTT LTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0025] M6 light chain variable region amino acid sequence (SEQ ID NO: 2): DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQRPDGTFKLLIYYKSRLQSGVPSRFSGSGSGIDYFLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0026] S2. CHO-K1 cell recovery, passaging and plasmid transfection 1. Cell Recovery and Passaging 1) Prepare 20 mL of basal medium in an E125 shake flask, add glutamine to a final concentration of 4 mM, and preheat in a 37°C incubator for 30 minutes. Simultaneously, prepare a 15 mL centrifuge tube and add 4 mL of fresh medium. 2) Remove the cells from the liquid nitrogen tank, thaw quickly in a 37°C water bath, transfer to a 15 mL centrifuge tube, blow well, and centrifuge at 1000 rpm for 5 minutes. 3) Discard the supernatant, resuspend the cells in 1 mL of fresh culture medium and transfer to an E125 shake flask. Take out a portion of the cell suspension and use an automatic cell counter to count the cell number and cell viability, which is generally 0.4-0.6×10 6 cells / mL cells, and then the flask was placed in a shaker for shaking culture (37°C, 5% CO2, 125 rpm); 4) Every other day, take out part of the cell suspension and use an automatic cell counter to count the cell number and cell viability. Then, subculture to 0.5×10 6 cells / mL, and the cells were passaged stably for 3 times.

[0027] 2. Plasmid Transfection 1) One day before plasmid transfection, passage the cells to 0.5×10 6 cells / mL to prepare plasmid transfection the next day; 2) Prepare 5 mL of fresh basal culture medium supplemented with 4 mM glutamine in a T25 flask and preheat in a 37°C incubator for 30 minutes. 3) Take out the cells and count them, then centrifuge them. The number of cells is 4×10 6 cells / mL, centrifuged at 1000 rpm for 5 min; 4) Discard the supernatant, resuspend with 5 mL of DPBS, and continue centrifugation at 1000 rpm for 5 minutes; 5) Discard the supernatant, resuspend in 100 μL electroporation buffer, add 1 mg of plasmid, and slowly pipette to mix well; 6) Neon electroporator: 1500-1700V / 15-25ms / 1 pulse; 7) Transfer the electroporated cell suspension into a T25 flask for static culture.

[0028] S3. Positive cell screening 1) 48 hours after electroporation, transfer the electroporated cells from the T25 flask into a 50 mL TPP shake tube, add MSX to a final concentration of 25 mM, and incubate in a shaker at 37°C. 2) For the first 5 days, add fresh culture medium every other day, about 2 mL each time. For the next 5 days, change the medium every other day by half centrifugation, resuspend in the same volume, pressurize with 25 mM MSX, and use an automatic cell counter to record the cell number and viability. After about 2 days, the cells will proliferate normally, and positive cells stably transfected with the plasmid will be obtained. The results of positive cell screening and recovery are shown in Table 1: Table 1 Positive cell screening recovery results

[0029] S4. Preparation and screening of high-expressing stable single-cell clones 1) After the cells have recovered, remove them and count them. Dilute them with monoclonal culture medium and plate them on a 96-well plate at a rate of 1 cell per well. Add 100 μL of monoclonal culture medium to each well. Select 10 96-well plates and incubate them in a 37°C incubator. After 2 hours, observe the 96-well plates under a microscope to identify wells with single cells and mark them. 2) After approximately 7 days, add 50 μL of fresh monoclonal culture medium and continue static culture. 3) After another 7 days (or 5 days), the confluence of the well plate can reach about 70%. Use Elisa to screen the wells with higher OD450 values, about 48 of which are expanded to 24-well plates and continue static culture; 4) Approximately 2 days later, use Octet to detect cell expression in the 24-well plate. Select the top 10 cell lines and expand them into 50 mL TPP shake tubes. Stably passage them three times to obtain a stably passaged monoclonal cell line.

[0030] S5. Fed-batch cell culture 1) The initial seeding density is 2.5-3.5×10 6 cells / mL, preferably 3.0×10 6 cells / mL, the volume is 15-30 mL, preferably 20 mL in this embodiment, in an E125 shake flask, cultured on a shaker (37°C, 5% CO2, 125 rpm), and the data for Day 0 are used; 2) Afterwards, remove cells every other day to measure cell density, viability, and sugar content; 3) The number of days of feeding can be determined according to the cell number and viability. Finally, the strain with the highest expression level was purified using an AKTA purification instrument. The results are shown in Table 2 and Figure 2 .

[0031] Table 2 Results of fed-batch culture of the highest cell line

[0032] The above cells are derived from the QuaCell® CHO-K1Q cell line, which originated from ECACC. The R&D team uses patented technology to domesticate and screen CHO-K1 cells. The basal culture medium is AltairCHO® Medium, the monoclonal culture medium is QuaMono™ PlusCHO, and the feed medium is StarCHOTM Feed medium and CDFS36 super concentrated feed.

[0033] Example 2 In this example, based on Example 1, 1-2% trehalose, 0.05-0.1 mM sodium selenite, and 3-8 mM L-proline were added to the basal medium for S2 cell recovery. Experimental results showed that a combination of 1.5% trehalose, 0.05 mM sodium selenite, and 5 mM proline could increase the recovery rate to >99%. Specifically, in the S2 cell recovery step, follow the following procedures: Resuscitation fluid preparation: AltairCHO® Medium, 4 mM glutamine, 1.5% trehalose, 0.05 mM sodium selenite, 5 mM L-proline; Resuscitation procedures: Remove the frozen cells from liquid nitrogen and thaw them in a 37°C water bath for ≤60 seconds; Centrifuge and discard the supernatant (1000 rpm, 5 min); Resuspend the cells in the above recovery solution pre-cooled to 4°C (avoid temperature shock); Transfer directly into E125 shake flask and culture with shaking at 125 rpm.

[0034] Furthermore, a metabolic activator combination is added to the S3 positive cell screening, including 1-5 mM sodium butyrate and 0.5-2 mM valproic acid, to enhance the chromatin openness and integration efficiency of the exogenous gene.

[0035] Specifically: During the S3 positive cell screening stage (when MSX is pressurized), add the following to the culture medium simultaneously: Sodium butyrate (2 mM): dissolved in PBS, filter sterilized and added to the culture medium; Valproic acid (1 mM): Prepare a 100 mM stock solution in DMSO and dilute to the final concentration before use.

[0036] The cell growth data are shown in Table 3 below: Table 3 Cell growth data

[0037] MSX and metabolic activators were added simultaneously. After adding the metabolic activator combination, the cell viability recovered to over 85% on the 6th day of MSX screening (compared to 50% in the group without addition). After adding the metabolic activator, cell proliferation was accelerated, and the cell count on Day 10 reached 3.2×10 6 / mL (activity 98%).

[0038] In order to demonstrate the technical effect of the innovative point of the present invention, comparative examples are as follows: Traditional methods: 1. Cell Recovery Cryopreservation medium: Fetal bovine serum (FBS) containing 8-10% DMSO or commercial freezing medium (such as CryoStor®).

[0039] Resuscitation process: Thaw cells in a 37°C water bath; The supernatant containing DMSO was removed by centrifugation; Incubate for 24-48 hours (until cells attach / recover); Then switch to suspension shaking culture.

[0040] 2. Positive Cell Screening Screening pressure: Single concentration of MSX (25-50 μM) throughout the screening process, no metabolic activator; Procedure: Let the cells stand for 48 hours after transfection → Add MSX → Let the cells stand or culture with shaking throughout the whole process (no stage optimization).

[0041] 3. Fed-batch culture Seeding density: 0.5-1.5×10 6 cells / mL (lower than the high-density inoculation of the present invention); Feeding strategy: constant sugar culture (glucose maintained at 4-6 g / L) or simple feeding (such as adding only glucose / glutamine).

[0042] The comparative data are shown in Table 4 below: Table 4 Comparison data table

[0043] From the above results, it can be seen that the method of the present invention has a high activity rate, increased antibody yield, and a short cycle.

[0044] The data in Table 1 are for Example 1 without the addition of a metabolic activator. In Example 2, the addition of sodium butyrate / valproic acid increased the activity to 85% on Day 6 (compared to 50% in Table 1); screening was completed on Day 10 (compared to Day 12 in Table 1). Table 2 shows the data of Example 1. In Example 2, under the same culture days (Day 18), the expression level increased to 11.2±0.4 g / L due to the enhanced transcription efficiency by the metabolic activator.

[0045] It should be noted that, for the sake of simplicity, the aforementioned embodiments are described as a series of action combinations. However, those skilled in the art should be aware that the present invention is not limited by the order of the actions described, because according to the present invention, certain steps may be performed in other orders or simultaneously. Secondly, those skilled in the art should also be aware that the embodiments described in this specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present invention.

[0046] In the embodiments provided in this application, it should be understood that the disclosed devices can be implemented in other ways. For example, the device embodiments described above are merely schematic. For example, the division of the above-mentioned units is only a logical function division. In actual implementation, there may be other division methods, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or communication connection shown or discussed can be through some interfaces, and the indirect coupling or communication connection between devices or units can be in the form of telecommunications or other forms.

[0047] The units described above as separate components may or may not be physically separate, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of these units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also fall within the scope of protection of the present invention.

Claims

1. A method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies, characterized in that: The steps include: S1. Antibody plasmid construction: Connect the antibody light and heavy chain genes to a mammalian expression vector, transform, and extract to obtain the antibody plasmid; S2. Cell recovery, passaging, and plasmid transfection: After recovery, CHO cells were directly cultured with shaking and transfected with the obtained antibody plasmid by electroporation. S3. Positive cell screening: Apply screening pressure within 48 hours after transfection and perform pressure screening under shaking conditions; S4. Preparation and screening of high-expressing stable single-cell clones: Single-cell clones were prepared using the limiting dilution method, and high-expressing clones were obtained through high-throughput screening. S5. Fed-batch culture of cells: fed-batch culture of selected clones with an inoculation density of 2.5-3.5×10 6 cells / mL; S6. Verification of cell line stability of antibody expression: Verify the stability of cell line passages for at least 20 generations.

2. The method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies according to claim 1, characterized in that: The CHO cells are CHO-K1 cell lines.

3. The method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies according to claim 1, characterized in that: The S2. cell recovery, passage and plasmid transfection include the following steps: The CHO cells were taken out of the liquid nitrogen tank, thawed quickly in a 37°C water bath, centrifuged and the supernatant discarded, and resuspended in basal medium supplemented with 4 mM glutamine. The cells were counted and cultured with shaking; CHO cells were passaged every other day for a total of 3 passages; The cells were passaged to an appropriate density one day before transfection, and plasmid transfection was performed the next day using a Neon electroporator and static culture in a 5 mL volume in a T25 flask.

4. The method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies according to claim 3, characterized in that: Electroporation parameters: cell density 3-5×10 6 cells / mL, sample volume 100 μL, voltage 1500-1700 V, pulse duration 15-25 ms.

5. The method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies according to claim 3, characterized in that: The S3. positive cell screening comprises the following steps: After 48 h of static culture in T25 flasks, MSX was pressurized; Transfer the culture medium from the T25 flask to a 50 mL TPP shake tube for shaking culture. Add 1-3 mL of fresh culture medium every other day for the first 5 days while pressurizing. During the next 5 days, the cells were centrifuged and the medium was changed every 36 hours, and the cells were resuspended in the same volume and pressurized with MSX. The number, viability and cell diameter of the cells were recorded during the treatment. After 2 days, the cells proliferated normally and positive cells stably transfected with the plasmid were obtained.

6. The method for rapidly constructing a stably transfected CHO cell line with high expression of IgG antibodies according to claim 1, characterized in that: The S4 high-throughput screening includes: In the first stage, the expression level of clones in 96-well plates was detected by ELISA; In the second stage, Octet was used to detect the expression level after amplification in 24-well plates.

7. The method for rapidly constructing a stably transfected CHO cell line with high IgG antibody expression according to claim 1, wherein: The S5. cell fed batch culture is specifically as follows: 2.5-3.5×10 6 cells / mL, with a total volume of 15-30 mL, and feed medium was used for 12-14 days.

8. The method for rapidly constructing a stably transfected CHO cell line with high IgG antibody expression according to claim 1, wherein: The antibody comprises the heavy chain sequence shown in SEQ ID NO: 1 and the light chain sequence shown in SEQ ID NO:

2.

9. The method for rapidly constructing a stably transfected CHO cell line with high IgG antibody expression according to claim 1, wherein: The following are added to the basic culture medium for S2 cell recovery: 1-2% trehalose, 0.05-0.1 mM sodium selenite and 3-8 mM L-proline.

10. The method for rapidly constructing a stably transfected CHO cell line with high IgG antibody expression according to claim 7, wherein: The S3 positive cell screening includes adding a metabolic activator combination including 1-5 mM sodium butyrate and 0.5-2 mM valproic acid.

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