A three-plasmid single-screening marker system for constructing stable cell pools and application thereof

Abstract

This invention relates to a three-plasmid single-screening marker system for constructing stable cell pools based on transposon technology and its application. The first and second subunits of the target protein are constructed separately into two vectors, and linked to the N-terminal and C-terminal peptides of Puro using the IRES2 sequence and the Npu DnaE integrity peptide. The plasmids used are no larger than 7.5 kb, have a low construction difficulty, can be synthesized simultaneously, and require a maximum of one week. The screening principle is that puromycin, with the assistance of the integrity peptide, splits into two halves and links to the first and second subunits respectively. Only when both the first and second subunit genes are integrated into the host cell genome can a complete and functional puromycin be formed. Therefore, only one antibiotic is needed to select positive cells during cell pool screening. The method developed in this invention based on the three-plasmid single-screening marker system can obtain a stable cell pool within 4 weeks, significantly reducing the cell pool development time.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a three-plasmid single-screening marker system for constructing stable cell pools and its application. Background Technology

[0002] A stable cell pool is a population of cells that can stably express a target gene over a long period of time, obtained through the introduction of exogenous genes, genome integration, and selection and enrichment. Compared to transient transfection systems, stable cell pools can maintain stable expression of the target gene during continuous passage, and are therefore widely used in recombinant protein production, gene function research, drug screening, cell therapy, and biopharmaceuticals.

[0003] Stable expression of different types of recombinant proteins still faces varying degrees of technical challenges. For example, membrane proteins and structurally complex macromolecules often struggle to achieve high-level stable expression due to low folding efficiency, complex post-translational modifications, or cytotoxicity. Antibody proteins, typically composed of two polypeptide chains (heavy and light chains), require balanced transcription and translation between these chains for proper expression. Furthermore, their expression is closely related to protein folding, disulfide bond formation, and secretion pathways. Therefore, efficiently constructing stable antibody cell lines with high expression levels and good stability has become a key technical issue in antibody drug development and industrial production. Stable antibody cell lines are considered the "cornerstone of drug development." A highly efficient and stable cell line directly impacts the yield, product quality, and production cost of the target antibody, and can significantly accelerate the drug's market launch.

[0004] The most common methods for constructing antibody cell lines are lentiviral therapy and DNA transposon technology. Lentiviral therapy involves integrating the gene of the target antibody into the genome of a host cell using lentiviruses. The standard procedure includes target antibody gene optimization, vector construction, lentivirus packaging and purification, cell infection, cell pool screening, single-cell cloning and screening, etc. Based on the number of plasmids, this method can be divided into single-plasmid systems and dual-plasmid systems. Single-plasmid systems use a 2A peptide or IRES sequence to link the antibody light and heavy chains into a single chain, which is then packaged into a single lentivirus. Dual-plasmid systems package the light and heavy chains of the antibody into two separate lentiviruses, which are then mixed in a specific ratio before infecting host cells.

[0005] Transposons are ubiquitous in the genomes of organisms and are mobile DNA sequences that can move (jump) from one gene location to another using their encoded enzymes. As a gene editing method, three commonly used transposon systems are the PiggyBac system, the Sleeping Beauty system, and the Leap-In system. The construction of antibody cell lines using transposon systems typically employs a dual-plasmid system: one plasmid contains the gene for transposase, and the other plasmid combines the light and heavy chains of the antibody, with each end linked to an inverted terminal repeat (ITR) sequence that can be specifically recognized by the transposase. The latter plasmid is linearized and co-transfected into the host cell along with the former. Linearizing the plasmid before transfection not only improves the efficiency of integrating foreign genes into the genome within the expression cassette but also reduces plasmid backbone sequence residue, minimizes the transient expression effects of the plasmid, and shortens the selection time. However, linearization increases the complexity of vector preparation and can lead to problems such as incomplete DNA breaks, fluctuating enzyme digestion efficiency, and decreased open-strand DNA stability, thereby affecting transfection efficiency and disrupting the consistency of subsequent cell pools.

[0006] The lentiviral method for constructing antibody-stabilized cell pools, particularly the single-plasmid system, is challenging due to the large insert size, high construction difficulty, long development time, and high cost. Furthermore, this random integration gene-editing method suffers from poor genetic stability, leading to inconsistent quality across different batches of cell lines and a long development cycle, typically 6-8 months. The conventional single-plasmid transposon system for constructing antibody-stabilized cell pools generally involves a promoter-light chain-promoter-heavy chain-promoter-selection marker structure, requiring three consecutive clonings or enzyme ligation, followed by three sequencing runs. This method is costly, time-consuming, and complex. The cell pool construction cycle using this method is generally 2-2.5 months, and the cell line construction cycle is 3-3.5 months. The latter commonly uses the GS selection system for positive cell screening, which requires specific GS-deficient CHO cell lines; the culture medium also has special requirements, needing to be strictly glutamine-free.

[0007] In conclusion, developing efficient methods for constructing stable cell pools is of great significance for the production of proteins such as antibodies. Summary of the Invention

[0008] To address the shortcomings of existing technologies and practical needs, this invention provides a three-plasmid single-screening marker system for constructing stable cell pools and its application, aiming to achieve rapid and efficient construction of stable cell pools.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a three-plasmid single-screening marker system for constructing a stable cell pool, the three-plasmid single-screening marker system comprising a first plasmid, a second plasmid, and a third plasmid; The first plasmid contains an expression cassette for expressing the first subunit of the target protein. The expression cassette of the first plasmid contains a 5' ITR sequence, a promoter, a coding sequence for the first subunit of the target protein, an IRES2 sequence, a coding sequence for the C-terminal peptide of Npu DnaE, ​​a coding sequence for the C-terminal peptide of puromycin, and a 3' ITR sequence. The second plasmid contains an expression cassette for expressing the second subunit of the target protein. The expression cassette of the second plasmid contains a 5' ITR sequence, a promoter, a coding sequence for the second subunit of the target protein, an IRES2 sequence, a coding sequence for the N-terminal peptide of puromycin, a coding sequence for the N-terminal peptide of Npu DnaE, ​​and a 3' ITR sequence. The third plasmid contains an expression cassette for expressing the transposase, and the expression cassette of the third plasmid contains the coding sequences for the promoter and the transposase.

[0010] This invention develops a three-plasmid single-screening marker system using Npu DnaE fission-type introns and puromycin as screening markers to construct stable cell pools. This system can be used to prepare target proteins composed of multiple subunits, such as antibodies. Specifically, when the target protein is an antibody, the first or second subunit can be the light or heavy chain of the target antibody.

[0011] Optionally, the initial plasmids of the first plasmid, the second plasmid, and the third plasmid each independently include any one of the following: pcDNA3.1 plasmid, pcDNA3.4 plasmid, pcDNA4 plasmid, pTT5 plasmid, pBI-CMV1 plasmid, pCMV-HA plasmid, pCMV6 plasmid, or pTetOne plasmid.

[0012] Optionally, the promoters in the first, second, and third plasmids may each independently include any one of the following: CMV promoter, SV40 promoter, EF1alpha promoter, CAG promoter, GAL1 promoter, or chicken beta-actin promoter.

[0013] Optionally, the transposase includes SB transposase or PB transposase.

[0014] Optionally, the C-terminal peptide of the Npu DnaE has a length of 37 aa, and the N-terminal peptide of the Npu DnaE has a length of 102 aa.

[0015] Optionally, the puromycin is divided into an N-terminal peptide and a C-terminal peptide between any two adjacent amino acid sites from position 76 to 85 (starting from the N-terminus); Optionally, the puromycin may be divided into an N-terminal peptide and a C-terminal peptide from positions 76-77, 78-79, 80-81, 82-83, or 84-85.

[0016] In one specific embodiment of the present invention, the puromycin is divided into an N-terminal peptide and a C-terminal peptide from position 84-85, wherein the length of the N-terminal peptide of the puromycin is 84 aa and the length of the C-terminal peptide of the puromycin is 115 aa.

[0017] It is understood that in this invention, "C-terminal peptide" and "N-terminal peptide" refer to dividing a protein into two parts, the N-terminus and the C-terminus, at a specific location.

[0018] Optionally, the amino acid sequence of the N-terminal peptide of the Npu DnaE includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the C-terminal peptide of the Npu DnaE includes the sequence shown in SEQ ID NO.2.

[0019] Optionally, the amino acid sequence of the N-terminal peptide of the puromycin includes the sequence shown in SEQ ID NO.3, and the amino acid sequence of the C-terminal peptide of the puromycin includes the sequence shown in SEQ ID NO.4.

[0020] Optionally, the nucleic acid sequence of the 5' ITR sequence includes the sequence shown in SEQ ID NO.5 or SEQ ID NO.7, and the nucleic acid sequence of the 3' ITR sequence includes the sequence shown in SEQ ID NO.6 or SEQ ID NO.8.

[0021] Optionally, the nucleic acid sequence of the IRES2 sequence includes the sequence shown in SEQ ID NO.9.

[0022] In a second aspect, the present invention provides the application of the three-plasmid single-screening marker system for constructing stable cell pools as described in the first aspect in the construction of stable cell pools.

[0023] Thirdly, the present invention provides a method for constructing a stable cell pool, the method comprising: The three-plasmid single-screening marker system for constructing a stable cell pool described in the first aspect is used to transfect host cells, and none of the three plasmids need to be linearized before they can be used for transfection; the transfected host cells are seeded in a culture medium containing puromycin for screening to obtain a stable cell pool.

[0024] Based on the three-plasmid system described in the first aspect, this invention establishes a method for constructing stable cell pools using plasmids with circular supercoiled double-stranded structures without requiring plasmid linearization. This method simplifies vector handling procedures, reduces degradation and mechanical damage during DNA processing, and improves the stability and reproducibility of plasmid preparation while ensuring transposition efficiency. Furthermore, supercoiled plasmids typically exhibit better cell transfection performance, contributing to increased positive cell yield and target protein expression levels. This method not only shortens the construction cycle of stable cell pools but also reduces the complexity of the process, providing a more convenient and reliable technical solution for establishing efficient and stable expression systems for target proteins.

[0025] Optionally, the host cells include Expi293F cells or CHO-K1 cells.

[0026] Optionally, the transfection method includes chemical transfection or electrotransfection.

[0027] Optionally, the screening method includes the following steps: (1) First round of pressure: 40-50 h after transfection, add 5-700 μg / mL of puromycin (e.g., 10, 20, 30, 40, 50, 60, 70, 100, 150, 200, 300, 400, 500, 600, 650, 670, 680 or 690 μg / mL, etc.) to the cells and continue culturing; (2) First recovery: After 30-40 h, the cells were centrifuged and cultured in fresh medium without puromycin; (3) Second round of pressurization: After 6-10 days, add 5-700 μg / mL of puromycin again for screening (e.g., 10, 20, 30, 40, 50, 60, 70, 100, 150, 200, 300, 400, 500, 600, 650, 670, 680 or 690 μg / mL, etc.). (4) Second recovery: After 30-40 h, the cells were centrifuged and cultured in fresh medium without puromycin; (5) Third round of pressurization: After 6-7 days, add 5-70 μg / mL of puromycin again for screening (e.g., 10, 20, 30, 40, 50, 60, 65, 67, 68 or 69 μg / mL, etc.). (6) Third round of recovery: After 3-4 days, the cell viability is maintained above 85% in a culture medium containing 5-70 μg / mL of puromycin (e.g., 10, 20, 30, 40, 50, 60, 65, 67, 68 or 69 μg / mL).

[0028] Compared with the prior art, the present invention has at least the following beneficial effects: This invention constructs a three-plasmid system, where the first and second subunits of the target protein are constructed on two separate vectors. Using the IRES2 sequence and the Npu DnaE integrin, these subunits are linked to the N-terminal and C-terminal fragments of puromycin, respectively. The plasmids used are no larger than 7.5 kb in size. The construction of the three plasmids is relatively easy and can be synthesized simultaneously in a short time, requiring a maximum of only one week. The screening principle is as follows: with the assistance of the integrin, puromycin is split into two parts, which are then linked to the first and second subunits, respectively. Only when both the first and second subunit genes are integrated into the host cell genome can a complete and functional puromycin be formed. Therefore, only one antibiotic is needed to screen for positive cells during cell pool screening. This invention, based on a three-plasmid single-screening marker system, can obtain a stable cell pool within four weeks, significantly shortening the cell pool development time. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the principle of using fissile puromycin as a cell pool screening marker.

[0030] Figure 2 The open reading frames (OPFs) of the three plasmids in the three-plasmid system of the antibody-stabilized cell pool constructed using the Sleeping Beauty transposon system are shown in order: the open reading frame of the antibody light chain expression plasmid, the open reading frame of the antibody heavy chain expression plasmid, and the open reading frame of the transposase expression plasmid.

[0031] Figure 3 This is a map of the first plasmid in a three-plasmid system of an antibody-stabilized cell pool constructed using the Sleeping Beauty transposon system, which contains the antibody light chain.

[0032] Figure 4 This is a map of the second plasmid in a three-plasmid system for an antibody-stabilized cell pool constructed using the Sleeping Beauty transposon system, which contains the antibody heavy chain.

[0033] Figure 5 This is a map of the third plasmid in the three-plasmid system of an antibody-stabilized cell pool constructed using the Sleeping Beauty transposase system, which contains the SB transposase.

[0034] Figure 6 The open reading frames (OPFs) of the three plasmids in the three-plasmid system of the antibody-stabilized cell pool constructed using the Piggy Bac transposon system are shown as the open reading frames of the antibody light chain expression plasmid, the antibody heavy chain expression plasmid, and the transposase expression plasmid, respectively.

[0035] Figure 7 This is a map of the first plasmid in a three-plasmid system of an antibody-stabilized cell pool constructed using the Piggy Bac transposon system, which contains the antibody light chain.

[0036] Figure 8 This is a map of the second plasmid in the three-plasmid system of an antibody-stabilized cell pool constructed using the Piggy Bac transposon system, which contains the antibody heavy chain.

[0037] Figure 9 This is a map of the third plasmid in the three-plasmid system of an antibody-stabilized cell pool constructed using the Piggy Bac transposase system, which contains the PB transposase.

[0038] Figure 10 The figures show the expression results of the anti-HER2 monoclonal antibody Expi293F cell pool constructed based on a three-plasmid single-screen marker system and SB transposon chemical transfection. Figure A shows the SDS-PAGE results of the antibody obtained after 5 days of batch culture at 37°C using protein A packing material from 40 mL of this cell pool. Figure B shows the HPLC-SEC results of the purified antibody. Figure C shows the growth curve and viability graph of this cell pool.

[0039] Figure 11 The figures show the expression results of the anti-HER2 monoclonal antibody Expi293F cell pool constructed based on a three-plasmid single-screen marker system and PB transposon chemical transfection method. Figure A shows the SDS-PAGE results of the antibody obtained after 5 days of batch culture at 37°C using protein A packing material from 40 mL of this cell pool. Figure B shows the HPLC-SEC results of the purified antibody. Figure C shows the growth curve and viability graph of this cell pool.

[0040] Figure 12 The images show the expression results of the anti-HER2 monoclonal antibody CHO-K1 cell pool constructed based on a three-plasmid single-screen marker system and SB transposon chemical transfection method. Figure A shows the SDS-PAGE results of the antibody obtained after 40 mL of the cell pool was cultured in batches at 37°C for 5 days and purified by protein A packing material. Figure B shows the HPLC-SEC results of the purified antibody. Figure C shows the growth curve and viability graph of the cell pool.

[0041] Figure 13The images show the expression results of the anti-HER2 monoclonal antibody CHO-K1 cell pool constructed using a three-plasmid single-screen marker system and PB transposon chemical transfection. Figure A shows the SDS-PAGE results of the antibody obtained after 5 days of batch culture at 37°C using protein A packing material from 40 mL of this cell pool. Figure B shows the HPLC-SEC results of the purified antibody. Figure C shows the growth curve and viability graph of this cell pool.

[0042] Figure 14 The images show the expression results of the anti-HER2 monoclonal antibody CHO-K1 cell pool constructed using a three-plasmid single-screen marker system and SB transposons via electroporation. Figure A shows the SDS-PAGE results of the antibody obtained after 5 days of batch culture at 37°C using protein A packing material from 40 mL of this cell pool. Figure B shows the HPLC-SEC results of the purified antibody. Figure C shows the growth curve and viability graph of this cell pool.

[0043] Figure 15 The images show the expression results of the anti-HER2 monoclonal antibody CHO-K1 cell pool constructed using a three-plasmid single-screen marker system and PB transposons via electroporation. Figure A shows the SDS-PAGE results of the antibody obtained after 5 days of batch culture at 37°C using protein A packing material from 40 mL of this cell pool. Figure B shows the HPLC-SEC results of the purified antibody. Figure C shows the growth curve and viability graph of this cell pool. Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0045] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0046] This invention develops a three-plasmid single-selection marker system and further develops a method for rapidly constructing stable cell pools. This method can obtain cell pools stably expressing the target protein after three pressure screenings within 4 weeks. Specifically, the selection marker, puromycin (Puro), is divided into an N-terminal peptide and a C-terminal peptide (named PuroN and PuroC, respectively) between any two adjacent amino acid sites from position 76 to 85. The N-terminal peptide of Npu DnaE (Npu DnaEN) contains 102 amino acids (amino acid sequence as shown in SEQ ID NO.1), and the C-terminal peptide (NpuDnaEC) contains 37 amino acids (amino acid sequence as shown in SEQ ID NO.2). The N-terminus of Puro contains amino acids 1-84 (amino acid sequence as shown in SEQ ID NO.3), or amino acids 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83, or 1-85, followed by the N-terminal peptide of Npu DnaE. An open reading frame (ORF) containing the second subunit gene of the target protein is added before the N-terminal peptide. Finally, the necessary ITR sequences for the transposition system (nucleic acid sequences as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8) are added to both ends. The first plasmid consists of amino acids 85-199 (amino acid sequence as shown in SEQ ID NO.4), 77-199, 78-199, 79-199, 80-199, 81-199, 82-199, 83-199, or 86-199, directly linked to the C-terminal peptide of Npu DnaE. An ORF containing the first subunit gene of the target protein is then added. Similarly, the necessary ITR sequences for the transposition system are added to both ends to form the second plasmid. The third plasmid expresses the transposase corresponding to the transposition system.

[0047] The principle of screening after co-transfecting the three plasmid systems into the host cell is illustrated in the diagram below. Figure 1 As shown, the Npu DnaE inteins retain high levels of protein trans-splicing activity at 37°C. Puro can utilize the two parts of this type of inteins, express them in cells, and then recombine them into a single protein with complete function. Through the action of transposons and Puro, cells in which both the first and second subunits of the target protein are integrated into the host cell genome can be selected.

[0048] In this embodiment of the invention, the construction of an antibody-stabilized cell pool is used as an example to verify the technical solution of the present invention.

[0049] Example 1 This embodiment describes the construction of a three-plasmid system.

[0050] Taking the construction of a three-plasmid system for anti-HER2 monoclonal antibodies (e.g., trastuzumab (Trast) as an example), it should be noted that the entire construction and validation process in this embodiment is only used to demonstrate that the three-plasmid single-screening system can rapidly construct a stable cell pool. The obtained anti-HER2 monoclonal antibodies are for basic scientific research only and not for any commercial purpose. The antibody light chain expression plasmid, antibody heavy chain expression plasmid, and transposase expression plasmid all use pcDNA3.1 as the starting plasmid and are synthesized de novo. The corresponding sequences are ligated into the pcDNA3.1 vector using enzyme digestion and ligation. A schematic diagram of the plasmid system structure is shown below. Figures 2-5 and Figures 6-9 As shown.

[0051] Figure 2 The first row contains the expression frames of the antibody light chain expression plasmid (SB_Light chain & PuroC), whose expression structures mainly include the 5' ITR sequence, CMV enhancer, CMV promoter, anti-HER2 monoclonal antibody light chain, IRES2 sequence (SEQ ID NO.9), C-terminal peptide of Npu DnaE, ​​C-terminal peptide of Puro, and 3' ITR sequence. The second row contains the expression frames of the antibody heavy chain expression plasmid (SB_Heavy chain & PuroN), whose expression structures mainly include the 5' ITR sequence, CMV enhancer, CMV promoter, anti-HER2 monoclonal antibody heavy chain, IRES2 sequence (SEQ ID NO.9), N-terminal peptide of Puro, N-terminal peptide of Npu DnaE, ​​and 3' ITR sequence. The third row contains the expression frames of the transposase expression plasmid (SBtransposase), whose expression structures mainly include the CMV enhancer... Enhancer, CMV promoter, and Sleeping Beauty (SB) transposase sequence, etc. Figure 3 Plasmid map of the first plasmid constructed based on the pcDNA3.1 vector (pcDNA) TM 3.1_SB_Light Chain & PuroC), with a total length of 5,739bp. Figure 4 Plasmid map of the second plasmid constructed based on the pcDNA3.1 vector (pcDNA) TM 3.1_SB_Heavy chain& PuroN), with a total length of 6,549bp. Figure 5 A plasmid map of the third plasmid constructed based on the pcDNA3.1 vector (pcDNA) TM 3.1_SB_transposase), with a total length of 6,446 bp.

[0052] Figure 6 The first row contains the expression frames of the antibody light chain expression plasmid (PB_Light chain & PuroC), which mainly include the 5' ITR sequence, CMV enhancer, CMV promoter, anti-HER2 monoclonal antibody light chain, IRES2 sequence, C-terminal peptide of Npu DnaE, ​​C-terminal peptide of Puro, and 3' ITR sequence. The second row contains the expression frames of the antibody heavy chain expression plasmid (PB_Heavy chain & PuroN), which mainly include the 5' ITR sequence, CMV enhancer, CMV promoter, anti-HER2 monoclonal antibody heavy chain, IRES2 sequence, N-terminal peptide of Puro, N-terminal peptide of Npu DnaE, ​​and 3' ITR sequence. The third row contains the expression frames of the transposase expression plasmid (PB transposase), which mainly include the CMV enhancer, CMV promoter, and Piggy. Bac (PB) transposase sequence, etc. Figure 7 Plasmid map of the first plasmid constructed based on the pcDNA3.1 vector (pcDNA) TM 3.1_PB_Light Chain & PuroC), with a total length of 5,824bp. Figure 8 Plasmid map of the second plasmid constructed based on the pcDNA3.1 vector (pcDNA) TM 3.1_PB_Heavy chain & PuroN), with a total length of 6,634bp. Figure 9 A plasmid map of the third plasmid constructed based on the pcDNA3.1 vector (pcDNA) TM 3.1_PB_transposase), with a total length of 7,211 bp.

[0053] Example 2 This embodiment utilizes the plasmid system constructed in Example 1 to construct an antibody cell pool.

[0054] 1. Cell transfection 1.1 Chemical transfection with Expi293F.

[0055] 1.1.1 Take 50 mL of 1.5-2×10 6 The Expi293F cells, with a viability of over 98%, were placed in a 125 mL Erlenmeyer flask.

[0056] 1.1.2 Mix the light chain plasmid, heavy chain plasmid and transposase plasmid in a ratio of 2:3:1 until homogeneous, and add 100 μg of plasmid to 1 mL of culture medium.

[0057] 1.1.3 Add 150 μL of 1 mg / mL PEI 25K to 1 mL of culture medium and mix well.

[0058] 1.1.4 Add the PEI from 1.1.3 to the plasmid from 1.1.2, mix well, and let stand at room temperature for 15 min.

[0059] 1.1.5 Add the mixture from 1.1.4 to 50 mL of cells from 1.1.1 and incubate in a shaker at 37°C, 8% CO2, and 125 rpm for 48 h.

[0060] 1.2 CHO-K1 chemical transfection 1.2.1 Take 20 mL of 5×10 6 CHO-K1 cells with a viability of 98% were placed in 125 mL Erlenmeyer flasks.

[0061] 1.2.2 Mix the light chain plasmid, heavy chain plasmid and transposase plasmid in a ratio of 2:3:1 and take 60 μg into a 1.5 mL centrifuge tube.

[0062] 1.2.3 Add 40 μL of TransZ to the plasmid and mix quickly and thoroughly.

[0063] 1.2.4 Add 140 μL of Trans8 to a centrifuge tube and mix quickly and thoroughly.

[0064] 1.2.5 Add the above mixture dropwise to the cells in 1.2.1 and incubate in a shaker at 37°C, 8% CO2, and 125 rpm for 48 h.

[0065] 1.3 Electroporation of CHO-K1 cells 1.3.1 Mix the light chain plasmid, heavy chain plasmid, and transposase plasmid in a ratio of 2:3:1 until homogeneous.

[0066] 1.3.2 Take 5 × 10 6 20 mL of CHO-K1 cells in the logarithmic growth phase were centrifuged and resuspended in 800 μL of electroporation buffer. Then, 200 μg of the plasmid mixed in 1.3.1 was added, and the cells were transferred to an electroporation cuvette.

[0067] 1.3.3 Select the preset CHO-K1 electro-spinning program for electro-spinning.

[0068] 1.3.4 Let stand at room temperature for 10 min.

[0069] 1.3.5 Aspirate the cells from the electroporation cup using 1 mL of preheated culture medium and transfer them to an Erlenmeyer flask containing 18 mL of culture medium. Incubate in a shaker at 37°C, 8% CO2, and 125 rpm for 48 h.

[0070] 1.4. Antibody cell pool screening.

[0071] 1.4.1 Screening of Expi293F cell pool 1.4.1.1 First round of pressurization: 48 h after transfection, add 10 μg / mL Puro to the cells and continue culturing.

[0072] 1.4.1.2 First round of recovery: After 32 h, when the cell viability dropped below 50%, the cells were centrifuged and cultured in fresh culture medium without Puro.

[0073] 1.4.1.3 Second round of pressurization: After 6-10 days, when the cell viability recovered to over 85%, 10 μg / mL uro was added again for screening.

[0074] 1.4.1.4 Second round of recovery: After 32 h, when the cell viability dropped to more than 50%, the cells were centrifuged and cultured in fresh medium without Puro.

[0075] 1.4.1.5 Third round of pressurization: After 4-6 days, when the cell viability recovered to over 85%, 10 μg / mL uro was added again for screening.

[0076] 1.4.1.6 Third round of recovery: After 3-5 days, the cell viability was maintained above 85% in culture medium containing 10 μg / mL Puro.

[0077] 1.4.2 CHO-K1 cell pool screening.

[0078] 1.4.2.1 First round of pressurization: 48 h after transfection, add 60 μg / mL Puro to the cells and continue culturing.

[0079] 1.4.2.2 First round of recovery: After 32 h, when the cell viability drops below 50%, the cells are centrifuged and cultured in fresh culture medium without Puro.

[0080] 1.4.2.3 Second round of pressurization: After 6-10 days, when the cell viability recovered to over 85%, 50 μg / mL uro was added again for screening.

[0081] 1.4.2.4 Second round of recovery: After 32 h, when the cell viability dropped to more than 50%, the cells were centrifuged and cultured in fresh culture medium without Puro.

[0082] 1.4.2.5 Third round of pressurization: After 6-7 days, when the cell viability recovered to over 85%, 60 μg / mL uro was added again for screening.

[0083] 1.4.2.6 Third round of recovery: After 3-4 days, the cell viability was maintained above 85% in culture medium containing 60 μg / mL Puro.

[0084] 1.5 Measurement of cell pool growth curves.

[0085] 1.5.1 Take cells from the antibody-stabilized cell pool constructed in 1.2 to achieve a final cell density of 1×10⁻⁶. 5 The volume is 10 mL, placed in a 125 mL Erlenmeyer flask containing cells. 60 μg / mL of Puro should be added to the cells.

[0086] 1.5.2 The cells from 1.5.1 were cultured in a shaker at 37°C, 8% CO2, and 25 rpm.

[0087] 1.5.3 Perform cell counting daily and record the two parameters: cell density and cell viability.

[0088] 1.5.4 When the cell viability is below 20%, stop cell counting and pour the cells into a waste container containing 84 disinfectant.

[0089] 1.6 Validation of antibody expression in cell pools.

[0090] 1.6.1 Validation of expression in cell pools.

[0091] 1.6.1.1 Take 40 mL of a solution with a viability of ≥98% and a density of 1.5 × 10⁻⁶. 6Cells were cultured in 125 mL Erlenmeyer flasks at 37°C, 8% CO2, and 125 rpm for 5 days.

[0092] 1.6.1.2 Centrifuge the cells at 5000 rpm for 15 min, and filter the supernatant sequentially through 0.45 μm and 0.22 μm filters.

[0093] 1.6.1.3 Add the supernatant to a purification column containing 1 mL of protein A packing material, collect the flow, and add it back to the purification column.

[0094] 1.6.1.4 Repeat the operation in 1.6.1.3 once.

[0095] 1.6.1.5 Add 50 mL of 1×PBS to clean the packing material.

[0096] 1.6.1.6 Elute with 15 mL of 0.1 M pH 3.0 citric acid and add 1 M pH 8.0 Tris-HCl buffer to the eluent.

[0097] 1.6.1.7 Concentrate the eluent, measure the concentration using a nanodropper, and run an SDS-PAGE gel.

[0098] The expression results of the anti-HER2 monoclonal antibody Expi293F cell pool constructed based on the three-plasmid single-selection marker system and SB transposons are as follows: Figure 10 As shown in the figure, Figure A is the SDS-PAGE result of the purified antibody. R represents the protein sample with added reducing loading buffer (abbreviation for Reduced), and NR represents the protein sample with added non-reducing loading buffer (abbreviation for Non-reduced). The results show that the Expi293F stable cell pool expressing anti-HER2 monoclonal antibody was successfully constructed by chemical transfection using SB transposons and the three-plasmid system of this invention. Figure B is the HPLC-SEC quality control result of the purified antibody. Combined with the gel image in Figure A, this result shows that the purified antibody has the correct conformation. Figure C is the growth curve of the stable cell pool in medium containing 10 μg / mL Puro, which shows that the cell pool can grow normally under the condition of 10 μg / mL Puro.

[0099] The expression results of the anti-HER2 monoclonal antibody Expi293F cell pool constructed based on the three-plasmid single-selection marker system and PB transposon are as follows: Figure 11As shown in the figure, Figure A is the SDS-PAGE result of the purified antibody. R represents the protein sample with added reducing loading buffer (abbreviation for Reduced), and NR represents the protein sample with added non-reducing loading buffer (abbreviation for Non-reduced). The results show that the Expi293F stable cell pool expressing anti-HER2 monoclonal antibody was successfully constructed by chemical transfection using PB transposons and the three-plasmid system of this invention. Figure B is the HPLC-SEC quality control result of the purified antibody. Combined with the gel image in Figure A, this result shows that the purified antibody has the correct conformation. Figure C is the growth curve of the stable cell pool in medium containing 10 μg / mL Puro, which shows that the cell pool can grow normally under the condition of 10 μg / mL Puro.

[0100] The expression results of the anti-HER2 monoclonal antibody CHO-K1 cell pool constructed based on the three-plasmid single-selection marker system and SB transposon chemical transfection method are as follows: Figure 12 As shown in the figure, Figure A is the SDS-PAGE result of the purified antibody, where R represents the protein sample with added reducing loading buffer (abbreviation for Reduced) and NR represents the protein sample with added non-reducing loading buffer (abbreviation for Non-reduced). The results indicate that a stable CHO-K1 cell pool expressing anti-HER2 monoclonal antibody was successfully constructed by chemical transfection using SB transposons and the three-plasmid system of this invention. Figure B is the HPLC-SEC quality control result of the purified antibody. Combined with the gel image in Figure A, this result indicates that the purified antibody has the correct conformation. Figure C is the growth curve of the stable cell pool in a medium containing 60 μg / mL Puro, indicating that the cell pool can grow normally under the condition of 60 μg / mL Puro.

[0101] The expression results of the anti-HER2 monoclonal antibody CHO-K1 cell pool constructed based on a three-plasmid single-selection marker system and PB transposon chemical transfection method are as follows: Figure 13As shown in the figure, Figure A is the SDS-PAGE result of the purified antibody. R represents the protein sample with added reducing loading buffer (abbreviation for Reduced), and NR represents the protein sample with added non-reducing loading buffer (abbreviation for Non-reduced). The results show that a stable CHO-K1 cell pool expressing anti-HER2 monoclonal antibody was successfully constructed by chemical transfection using PB transposons and the three-plasmid system of this invention. Figure B is the HPLC-SEC quality control result of the purified antibody. Combined with the gel image in Figure A, this result shows that the purified antibody has the correct conformation. Figure C is the growth curve of the stable cell pool in medium containing 60 μg / mL Puro, which shows that the cell pool can grow normally under the condition of 60 μg / mL Puro.

[0102] The expression results of the anti-HER2 monoclonal antibody CHO-K1 cell pool constructed based on the three-plasmid single-selection marker system and SB transposon electroporation transfection method are as follows: Figure 14 As shown in the figure, Figure A is the SDS-PAGE result of the purified antibody. R represents the protein sample with added reducing loading buffer (abbreviation for Reduced), and NR represents the protein sample with added non-reducing loading buffer (abbreviation for Non-reduced). The results show that a stable CHO-K1 cell pool expressing anti-HER2 monoclonal antibody was successfully constructed by electroporation using SB transposons and the three-plasmid system of this invention. Figure B is the HPLC-SEC quality control result of the purified antibody. Combined with the gel image in Figure A, this result shows that the purified antibody has the correct conformation. Figure C is the growth curve of the stable cell pool in a medium containing 60 μg / mL Puro, indicating that the cell pool can grow normally under the condition of 60 μg / mL Puro.

[0103] The expression results of the anti-HER2 monoclonal antibody CHO-K1 cell pool constructed based on the three-plasmid single-selection marker system and PB transposon electroporation transfection method are as follows: Figure 15As shown in the figure, Figure A is the SDS-PAGE result of the purified antibody. R represents the protein sample with added reducing loading buffer (abbreviation for Reduced), and NR represents the protein sample with added non-reducing loading buffer (abbreviation for Non-reduced). The results show that a stable CHO-K1 cell pool expressing anti-HER2 monoclonal antibody was successfully constructed by electroporation using the PB transposon and the three-plasmid system of this invention. Figure B is the HPLC-SEC quality control result of the purified antibody. Combined with the gel image in Figure A, this result shows that the purified antibody has the correct conformation. Figure C is the growth curve of the stable cell pool in a medium containing 60 μg / mL Puro, which shows that the cell pool can grow normally under the condition of 60 μg / mL Puro.

[0104] In summary, this invention relates to a three-plasmid single-screening marker system for constructing stable cell pools based on transposon technology and its applications. The three-plasmid system constructed in this invention involves constructing the first and second subunits of the target protein onto two donor vectors, respectively, and then linking them to the N-terminal and C-terminal fragments of puromycin under the influence of the IRES2 sequence and the Npu DnaE integrin, respectively; the transposase is constructed onto a separate vector. The size of the plasmids used does not exceed 7.5 kb, thus the construction of the three plasmids is relatively easy; they can also be synthesized simultaneously, with a maximum construction time of one week, which greatly shortens the plasmid synthesis cycle. The screening method used in this invention is as follows: under the influence of the integrin, puromycin is split into two fragments. The N-terminal fragment of the purine toxin is linked to the second subunit of the target protein via the IRES2 motif, and the C-terminal fragment of the purine toxin is linked to the first subunit of the target protein via the IRES2 motif. Only when both the light chain and heavy chain genes are integrated into the host cell genome can a complete and functional puromycin be formed under the protein trans-splicing action of the integrin. Therefore, this system requires only one antibiotic to effectively screen positive cells, avoiding the operational complexity and cell growth stress caused by multiple rounds of resistance screening and progressively pressurized culture in traditional multi-screening marker systems. Compared to conventional stable transfection systems, the three-plasmid single-screening marker system established in this invention optimizes vector design and screening strategies. It not only eliminates the need for linearization of donor plasmids but also achieves stable expression of the target gene while maintaining high transposition integration efficiency. This system reduces preliminary steps such as plasmid pretreatment, enzyme digestion purification, and quality control, lowering the risk of DNA degradation and transfection efficiency fluctuations. It also helps maintain high transfection activity of supercoiled plasmids, thereby improving the efficiency of obtaining positive cells and the consistency of the cell pool. Based on these optimizations, this system can complete the construction of a stable expression cell pool in approximately four weeks, significantly shortening the development cycle and improving cell line development efficiency compared to traditional methods. It provides a more efficient, convenient, and stable technical platform for subsequent target protein expression evaluation, process development, and stable cell line screening.

[0105] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A three-plasmid single-screening marker system for constructing stable cell pools, characterized in that, The three-plasmid single-screening marker system includes a first plasmid, a second plasmid, and a third plasmid; The first plasmid contains an expression cassette for expressing the first subunit of the target protein. The expression cassette of the first plasmid contains a 5' ITR sequence, a promoter, a coding sequence for the first subunit of the target protein, an IRES2 sequence, a coding sequence for the C-terminal peptide of Npu DnaE, ​​a coding sequence for the C-terminal peptide of puromycin, and a 3' ITR sequence. The second plasmid contains an expression cassette for expressing the second subunit of the target protein. The expression cassette of the second plasmid contains a 5' ITR sequence, a promoter, a coding sequence for the second subunit of the target protein, an IRES2 sequence, a coding sequence for the N-terminal peptide of puromycin, a coding sequence for the N-terminal peptide of Npu DnaE, ​​and a 3' ITR sequence. The third plasmid contains an expression cassette for expressing the transposase, and the expression cassette of the third plasmid contains the coding sequences for the promoter and the transposase.

2. The three-plasmid single-screening marker system for constructing stable cell pools according to claim 1, characterized in that, The initial plasmids of the first plasmid, the second plasmid, and the third plasmid each independently include any one of the following: pcDNA3.1 plasmid, pcDNA3.4 plasmid, pcDNA4 plasmid, pTT5 plasmid, pBI-CMV1 plasmid, pCMV-HA plasmid, pCMV6 plasmid, or pTetOne plasmid.

3. The three-plasmid single-screening marker system for constructing stable cell pools according to claim 1 or 2, characterized in that, The promoters in the first, second, and third plasmids each independently include any one of the following: CMV promoter, SV40 promoter, EF1alpha promoter, CAG promoter, GAL1 promoter, or chicken beta-actin promoter; Optionally, the transposase includes SB transposase or PB transposase.

4. The three-plasmid single-screening marker system for constructing stable cell pools according to any one of claims 1-3, characterized in that, The C-terminal peptide of the Npu DnaE has a length of 37 aa, and the N-terminal peptide of the Npu DnaE has a length of 102 aa.

5. The three-plasmid single-screening marker system for constructing stable cell pools according to any one of claims 1-4, characterized in that, The puromycin is divided into an N-terminal peptide and a C-terminal peptide between any two adjacent amino acid sites from position 76 to 85. Optionally, the puromycin may be divided into an N-terminal peptide and a C-terminal peptide from positions 76-77, 78-79, 80-81, 82-83, or 84-85.

6. The three-plasmid single-screening marker system for constructing stable cell pools according to any one of claims 1-5, characterized in that, The amino acid sequence of the N-terminal peptide of the Npu DnaE includes the sequence shown in SEQ ID NO.1, and the amino acid sequence of the C-terminal peptide of the Npu DnaE includes the sequence shown in SEQ ID NO.

2.

7. The three-plasmid single-screening marker system for constructing stable cell pools according to any one of claims 1-6, characterized in that, The amino acid sequence of the N-terminal peptide of the puromycin includes the sequence shown in SEQ ID NO.3, and the amino acid sequence of the C-terminal peptide of the puromycin includes the sequence shown in SEQ ID NO.

4.

8. The three-plasmid single-screening marker system for constructing stable cell pools according to any one of claims 1-7, characterized in that, The nucleic acid sequence of the 5' ITR sequence includes the sequences shown in SEQ ID NO.5 and SEQ ID NO.7, and the nucleic acid sequence of the 3' ITR sequence includes the sequences shown in SEQ ID NO.6 and SEQ ID NO.8; Optionally, the nucleic acid sequence of the IRES2 sequence includes the sequence shown in SEQ ID NO.

9.

9. The application of the three-plasmid single-screen marker system for constructing stable cell pools according to any one of claims 1-8 in constructing stable cell pools.

10. A method for constructing a stable cell pool, characterized in that, The method includes: The three-plasmid single-screening marker system for constructing a stable cell pool as described in any one of claims 1-8 is used to transfect host cells, wherein none of the three plasmids need to be linearized before they can be used directly for transfection; the transfected host cells are seeded in a culture medium containing puromycin for screening to obtain a stable cell pool.

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