Recombinant gene XBP1-BIP for constructing recombinant cell for improving antibody expression quantity, expression vector, recombinant cell for improving antibody expression quantity and application of recombinant cell
By designing the recombinant gene XBP1-BIP and constructing the expression vector XB-pLVX-IRES-puro, the problem of complexity and limited yield of the antibody expression system in mammalian cells is solved, and efficient and low-cost antibody expression and production are achieved.
Patent Information
- Application Number
- CN202411956136.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-28
- Publication Date
- 2025-05-09
AI Technical Summary
The current antibody expression system constructed based on mammalian cells has problems such as complex construction process, high culture cost, limited production of expression antibodies, and prone to genetic mutations, deletions, and rearrangements of the genome, which affects the yield and quality of antibodies.
By designing the recombinant gene XBP1-BIP and ligating it through IRES elements, the expression vector XB-pLVX-IRES-puro was constructed to construct recombinant cells 293-XB and increase the antibody expression level.
It has achieved efficient production of target antibodies in a short period of time, significantly increasing the expression of antibodies, reducing costs, and to a certain extent the potential biosafety risks caused by gene integration.
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Figure CN119955827A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of antibody technology, and in particular to a recombinant gene XBP1-BIP and an expression vector for constructing a recombinant cell with increased antibody expression, a recombinant cell with increased antibody expression, and applications thereof. Technical Background
[0002] The industrial manufacturing process of biopharmaceuticals is highly dependent on cell expression platforms to maximize production output and reduce related costs. Therefore, it is very necessary to develop and implement new strategies to achieve high-yield mammalian cell expression. The FreeStyle 293F cell line is prepared from a low-generation master cell bank culture derived from the parental 293-F cells (re-cloned by limiting dilution). As a mammalian host cell, the advantages of this cell are: (I) easy to transfect, with good tolerance and high transfection efficiency; (II) good growth characteristics and the ability to grow in suspension, which is very beneficial for large-scale industrial production; (III) the ability to produce very high levels of protein; (IV) the ability to grow rapidly and culture at high density in the culture system.
[0003] In the past decade, various successful transcriptional and translational engineering strategies have significantly boosted mammalian cell productivity. Currently, post-translational capacity limitations have been considered the main bottleneck that prevents mammalian cells from fully exploiting their physiological production capacity in biopharmaceutical production scenarios.
[0004] When B cells differentiate into plasma cells, they lose the expression of most of the B cell characteristics and undergo free radical recombination, which enables them to secrete large amounts of the protein immunoglobulin (Ig). There are two essential transcriptional regulators for plasma cell differentiation: the transcriptional repressor Blimp-1 encoded by the prdm1 gene and the a b-ZIP family transcriptional activator XBP1. XBP1 acts downstream of Blimp-1. XBP1s is a key factor in the inositol-requiring enzyme 1 (IRE1) / XBP1 signaling pathway in the endoplasmic reticulum UPR, and can induce the expression of molecular chaperones and related proteins in the endoplasmic reticulum protein degradation pathway. It can be seen that XBP1 is a key regulatory factor in the differentiation of B cells into plasma cells that secrete immunoglobulins, which can relieve endoplasmic reticulum stress by assisting the folding of unfolded proteins and degrading them.
[0005] Binding immunoglobulin protein (BIP), also known as HSPAS, is an important molecular chaperone located in the endoplasmie retieulum (ER). It is a member of the heat shock protein (Hsp70) family, also known as GRP78. It is an ER-resident protein that can bind to many incompletely folded and unassembled proteins. For many secreted or membrane-bound proteins, their binding to BIP is transient; while for proteins that are misfolded, improperly glycosylated or otherwise insufficiently secreted, their binding to BIP can be more stable. As an ER chaperone, BIP plays a key role in protein folding and quality control in the ER lumen.
[0006] The development of efficient bioprocessing strategies is crucial for the industrial production of recombinant proteins with therapeutic and preventive significance. Recombinant proteins used in the fields of therapeutic drugs, vaccines and diagnostic reagents are mainly produced in laboratory and large-scale environments using prokaryotic and eukaryotic expression host systems (such as mammalian cells, bacteria, yeast, insect cells and transgenic plants). For some recombinant protein drugs with complex structures and functions that depend on mammalian-specific post-translational modifications (such as correct glycosylation, phosphorylation, etc.), mammalian cell culture is the main production method. However, mammalian cell culture is expensive, has a long production cycle, and relatively high technical requirements. Therefore, in industrial production, screening out cell lines with high yield and strong stability can be used as host cells for the transient expression system of recombinant proteins, which can effectively increase the expression of antibodies while significantly reducing costs. This is extremely urgent and of great significance to enterprises.
[0007] FreeStyle 293 can be used for transient gene expression (TGE) due to its easy transfection. Compared with stable expression systems, transient expression systems can produce target proteins in a short time, which can greatly improve work efficiency and reduce costs; in addition, exogenous genes in transient expression systems generally will not be integrated into the genome of host cells, reducing the potential impact on the stability of the host cell genome, reducing the risk of cell carcinogenesis or other adverse phenotypes caused by gene integration, and improving the safety and reliability of the production process. Summary of the invention
[0008] The technical problem to be solved by the present invention is that the current antibody expression system constructed based on mammalian cells not only has a complex construction process, high culture cost, and limited expression of antibody production, but also in the process of long-term subculture, the genome is prone to genetic variations such as mutation, deletion, rearrangement, etc., which affect the production and quality of antibodies.
[0009] In order to solve the technical problem, the present invention provides a recombinant gene XBP1-BIP and an expression vector for constructing a recombinant cell for increasing the antibody expression amount, a recombinant cell for increasing the antibody expression amount and applications thereof.
[0010] The object of the present invention is achieved by: A recombinant gene XBP1-BIP for constructing a recombinant cell with increased antibody expression, wherein the recombinant gene XBP1-BIP is obtained by connecting codon-optimized XBP1 and BIP gene sequences through an IRES element; the nucleotide sequence of the recombinant gene XBP1-BIP is shown in SEQ ID No: 5.
[0011] An expression vector for constructing a recombinant cell for increasing the expression amount of an antibody, wherein the expression vector is obtained by cloning the nucleotide sequence shown in SEQ ID No: 5 into a plasmid vector pLVX-IRES-puro.
[0012] The method for constructing the expression vector comprises the following steps: (1) Synthesizing the target gene: The artificially synthesized nucleotide sequence is shown in SEQ ID No: 5; (2) Construction of gene expression vector: The nucleotide sequence shown in SEQ ID No: 5 was inserted into the plasmid vector pLVX-IRES-puro through the EcoR1 / XbaI restriction site to obtain the expression vector XB-pLVX-IRES-puro.
[0013] A recombinant cell for increasing the expression amount of an antibody, wherein the recombinant cell contains the expression vector according to claim 2.
[0014] A method for constructing a recombinant cell for increasing the expression of an antibody comprises the following steps: (1) Lentiviral packaging in HEK 293T adherent cells One day before transfection, HEK 293T cells were trypsinized and plated on a 6 cm plate at a density of 5 × 10^5 cells / ml in 5 ml of DMEM complete medium. On the day of transfection, when the cell confluence reached 80%-90%, the expression vector XB-pLVX-IRES-puro and the plasmids PSPAX2 and PMD2G used for packaging were integrated into HEK 293T cells using PEI transfection reagent at a volume ratio of 4:3:1. (2) Puromycin screening of cell lines After 48 h of transfection, the cells were centrifuged at 4000 rpm and 4°C for 10 min to collect the virus supernatant. FreeStyle 293F suspension cells were inoculated into 6-well plates at a cell inoculation volume of 1×106 cells / well; the virus solution was used to infect the FreeStyle 293F suspension cells that had been cultured in advance; 3 days after infection, pressure screening was performed with 2 µg / mL, 3 µg / mL, 4 µg / mL and 5 µg / mL of puromycin screening solution, respectively, and the solution was changed and the drug was added every 2-3 days to observe the cell growth state, and it was determined that the maximum drug resistance pressure of the cell line was about 4 µg / mL; 4 µg / mL Puromycin was used for pressure screening to obtain a stable cell pool; (3) Monoclonal cell line screening After the cells were stabilized, monoclonal cells were screened by limiting dilution method with 3 µg / mL Puromycin. After plating on 96-well plates, the monoclonal cell line was determined by monoclonal imaging to obtain the recombinant cell 293-XB that can simultaneously express XBP1 and BIP. The monoclonal cell line with the best growth state was determined through step-by-step screening using 24-well plates and 6-well plates, and was frozen in time for simultaneous result verification.
[0015] An application of a recombinant cell 293-XB for increasing the expression of an antibody, wherein the recombinant cell 293-XB is obtained by the method according to any one of claims 1 to 5, and the application comprises the following steps: (1) Select the target antibody and construct the PPT5 transient expression vector; (2) Use a plasmid extraction kit to extract plasmid DNA and purify the plasmid DNA; (3) 293-XB cells were revived using OPM-293 CD05 Medium. One day before antibody transfection, cells were cultured at 2×10 6 The cells were inoculated into fresh culture medium at a density of 3 × 10 cells / mL and cultured in a shaker at 37°C, 5% CO2, and 150 rpm. On the day of transfection, samples were taken to count the cell density and viability and the cell density was adjusted to 3 × 10 6 cells / mL, viability ≥95%; (4) The recombinant plasmid was transfected into the 293-XB cell line using PEI transfection reagent for expression. The specific transfection and expression process was as follows: plasmid DNA and PEI transfection reagent were diluted with OPM-293 CD05 Medium, and the diluted PEI transfection reagent was added to the diluted plasmid DNA at a ratio of plasmid DNA: PEI transfection reagent = 1:4; the complex was incubated at room temperature for about 20 minutes; 22 hours after transfection, 5% (v / v) 293-ProFeed was added to the shake flask. During the entire transient expression process, the glucose concentration was maintained above 4 g / L, and the cells were harvested when the cell viability was less than 70%; (5) The harvested target antibody was filtered through a 0.45 µm filter membrane and purified using a chromatography column filled with Protein A (GE) filler. The dissociation peak was collected and the protein content was detected using a spectrophotometer NanoDrop One and the protein purity was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE.
[0016] Compared with the prior art, the technical beneficial effects of the present invention are: providing a recombinant gene XBP1-BIP and an expression vector for constructing a recombinant cell with increased antibody expression, a recombinant cell with increased antibody expression and its application; the modified cell strain has high versatility and can be used as a host cell for a transient expression system of the target antibody; the cell strain constructed by this method can produce the target antibody in a short time, effectively increase the antibody expression amount, significantly reduce the cost, and reduce the potential biosafety risks caused by gene integration to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 1% agarose gel electrophoresis plots for XBP1 and BIP genes.
[0018] Figure 2 For 293-X, 293-B and 293-XB cell lines: Effect of different Puromycin concentrations on cell viability.
[0019] Figure 3 Comparison of the expression levels of MAb1-MAb6 antibodies in different cell lines.
[0020] Figure 4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis of MAb1 antibody (1 mg / mL) expressed in different cell lines. DETAILED DESCRIPTION
[0021] The present invention will be further described below in conjunction with specific embodiments.
[0022] 1. Biomaterials FreeStyle 293F cells were purchased from Thermo Fisher Scientific; HEK 293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences.
[0023] 2. Synthesize the target gene Based on the known XBP1 and BIP gene sequences published in GenBank, codon optimization was performed, that is, the genes were redesigned by avoiding rare codons, utilizing preferred codons, simplifying the secondary structure of mRNA, optimizing repetitive sequences, eliminating restriction enzyme sites, and adjusting the GC content to improve translation efficiency and thus improve protein expression levels.
[0024] The nucleotide sequence of the codon-optimized XBP1 gene is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID No: 2; the nucleotide sequence of the codon-optimized BIP gene is shown in SEQ ID No: 3, and the amino acid sequence is shown in SEQ ID No: 4; in addition, by inserting an IRES element, the genes of XBP1 and BIP are connected through the IRES element, named XBP1-BIP, and the sequence is shown in SEQ ID No: 5.
[0025] XBP1-BIP gene was synthesized by GenScript Biotechnology. Using it as a template, primers XBP1-F / XBP1-R and BIP-F / BIP-R (Table 1) were designed to amplify the target fragments XBP1 and BIP by PCR, respectively. The reaction procedure was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 45 s, 52℃ annealing for 45 s, and 72℃ extension for 2 min, for a total of 30 cycles; and finally 72℃ extension for 10 min. PCR results were verified by 1% agarose gel electrophoresis ( Figure 1 ).
[0026] Table 1 PCR amplification primer sequences 3. Construction of gene expression vector The target fragments XBP1 and BIP were inserted into the plasmid vector pLVX-IRES-puro (Ubao Biotech) through the EcoR1 / XbaI restriction site; the plasmids were named X-pLVX-IRES-puro and B-pLVX-IRES-puro. The XBP1-BIP sequence was sent to BGI for synthesis and also subcloned into pLVX-IRES-puro, named XB-pLVX-IRES-puro. The constructed X-pLVX-IRES-puro and B-pLVX-IRES-puro plasmids were verified by full-length Jinbiological sequencing, and the results showed that the sequences were correct.
[0027] 4. Lentiviral packaging in HEK 293T adherent cells One day before transfection, HEK 293T cells were trypsinized and plated on 6 cm dishes at a density of 5×10^5 cells / ml in 5 ml of DMEM complete medium. On the day of transfection, the cell confluence reached 80%-90%. The three expression vectors constructed above (X-pLVX-IRES-puro, B-pLVX-IRES-puro and XB-pLVX-IRES-puro) were combined with the packaging plasmids PSPAX2 and PMD2G at a volume ratio of 4:3:1 and integrated into HEK 293T cells using PEI transfection reagent (Polysciences, USA).
[0028] 5. Puromycin screening of cell lines After 48 h of transfection, the cells were centrifuged at 4000 rpm and 4°C for 10 min to collect the virus supernatant. FreeStyle 293F suspension cells were inoculated into 6-well plates at a cell inoculation volume of 1×10 6 cells / well; the virus solution was used to infect the FreeStyle 293F suspension cells that had been cultured in advance; 3 days after infection, pressure screening was performed with 2 µg / mL, 3 µg / mL, 4 µg / mL and 5 µg / mL of Puromycin screening solution, and the solution and drug were changed every 2-3 days to observe the cell growth state and determine the maximum drug resistance of the cell line; the results showed that at the 5 µg / mL Puromycin screening concentration, the cell viability decreased rapidly, and there was no recovery in the later stage, and death occurred. Under the 2 µg / mL Puromycin screening pressure, the cell viability began to gradually recover after 3-5 days of decline. Under the 3µg / mL Puromycin screening pressure, the cell viability recovery time was about one week, and under the 4 µg / mL Puromycin screening pressure, the cell recovery time was about 10 days. The comprehensive maximum drug resistance was determined to be about 4 µg / mL ( Figure 2 ). Stable cell pools were obtained by pressure screening using 4 µg / mL Puromycin.
[0029] VI. Monoclonal cell line screening After the cells were stabilized, monoclonal cells were screened by limiting dilution method with 3 µg / mL Puromycin. After plating on 96-well plates, monoclonal cell lines were identified by monoclonal imaging, including cell lines expressing XBP1 and BIP alone, and cell lines that could express XBP1 and BIP simultaneously, which were named 293-X, 293-B, and 293-XB, respectively. The monoclonal cell lines with the best growth status were determined through step-by-step screening of 24-well plates and 6-well plates, and were frozen in time and the results were verified simultaneously.
[0030] VII. Comparison of transient expression of target antibodies in different cell lines (1) Select the world's best-selling drugs: human-mouse chimeric antibodies (Remicade, Rituxan), humanized antibodies (Herceptin, Avastin), and fully human antibodies (Humira, Keytruda). Select two representative drugs for each antibody type and name them MAb1-MAb6, and construct the PPT5 transient expression vector; (2) Plasmid DNA was extracted and purified using a plasmid extraction kit (TransGen Biotech, Beijing, China); (3) FreeStyle293F, 293-X, 293-B, and 293-XB cell lines were revived using OPM-293 CD05 Medium (OPM, Shanghai, China). One day before antibody transfection, cells were cultured at 2 × 10 6 The cells were inoculated into fresh culture medium at a density of 3 × 10 cells / mL and cultured in a shaker at 37°C, 5% CO2, and 150 rpm. On the day of transfection, samples were taken to count the cell density and viability and the cell density was adjusted to 3 × 10 6 cells / mL, viability ≥95%; (4) The recombinant plasmid was transfected into FreeStyle 293F (control group), 293-X, 293-B and 293-XB cell lines using PEI transfection reagent for expression, so as to compare the effects of different cell lines on antibody expression. The specific transfection and expression process was as follows: plasmid DNA and PEI transfection reagent were diluted with OPM-293 CD05 Medium, and the diluted PEI transfection reagent was added to the diluted plasmid DNA at a ratio of DNA: PEI transfection reagent = 1:4; the complex was incubated at room temperature for about 20 minutes; 22 hours after transfection, 5% (v / v) 293-ProFeed was added to the shake flask, and the glucose concentration was maintained above 4 g / L during the entire transient expression process. The cells were harvested when the cell viability was less than 70%; (5) The harvested target antibody was filtered through a 0.45 µm filter membrane and purified using a chromatography column filled with Protein A (GE) filler. The dissociation peak was collected and the protein content was detected using a spectrophotometer NanoDrop One (Thermo Fisher Scientific, Shanghai, China). The protein purity was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
[0031] The results showed that the expression levels of 4 antibodies (MAb1, MAb3, MAb4, and MAb6) in the 293-XB cell line were significantly increased, and compared with the use of 293-X, 293-B, and FreeStyle 293F cells, it had a more obvious advantage; the expression level of MAb4 antibody in the three newly constructed cell lines (293-X, 293-B, and 293-XB) was higher than that of the conventional FreeStyle 293F, among which the 293-XB cell line performed better; the expression level of MAb5 antibody in several cell lines was not much different ( Figure 3 ). Moreover, the SDS-PAGE of antibodies expressed by different cell lines at the same concentration (1 mg / mL) showed little difference in purity. Taking MAb1 as an example, see Figure 4 .
[0032] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.
Claims
1. A recombinant gene XBP1-BIP for constructing a recombinant cell with increased antibody expression, characterized in that: The recombinant gene XBP1-BIP is obtained by connecting the codon-optimized gene sequences of XBP1 and BIP through an IRES element; the nucleotide sequence of the recombinant gene XBP1-BIP is shown in SEQ ID No:
5.
2. An expression vector for constructing a recombinant cell for increasing the expression amount of an antibody, characterized in that: The expression vector is obtained by cloning the nucleotide sequence shown in SEQ ID No: 5 into the plasmid vector pLVX-IRES-puro.
3. The method for constructing an expression vector according to claim 2, wherein: The following steps are involved: (1) Synthesizing the target gene: The artificially synthesized nucleotide sequence is shown in SEQ ID No: 5; (2) Construction of gene expression vector: The nucleotide sequence shown in SEQ ID No: 5 was inserted into the plasmid vector pLVX-IRES-puro through the EcoR1 / XbaI restriction site to obtain the expression vector XB-pLVX-IRES-puro.
4. A recombinant cell for increasing the expression of an antibody, characterized in that: The recombinant cell contains the expression vector according to claim 2.
5. The method for constructing a recombinant cell for increasing antibody expression according to claim 4, characterized in that: The following steps are involved: (1) Lentiviral packaging in HEK 293T adherent cells One day before transfection, HEK 293T cells were trypsinized and plated on a 6 cm plate at a density of 5×10^5 cells / ml in 5 ml of DMEM complete medium. On the day of transfection, when the cell confluence reached 80%-90%, the expression vector XB-pLVX-IRES-puro and the plasmids PSPAX2 and PMD2G used for packaging were integrated into HEK293T cells using PEI transfection reagent at a volume ratio of 4:3:
1. (2) Puromycin screening of cell lines After 48 h of transfection, the cells were centrifuged at 4000 rpm and 4°C for 10 min to collect the virus supernatant. FreeStyle 293F suspension cells were inoculated into 6-well plates at a cell inoculation volume of 1×10 6 cells / well; the virus solution was used to infect the FreeStyle293F suspension cells that had been cultured in advance; 3 days after infection, pressure screening was performed with 2 µg / mL, 3 µg / mL, 4 µg / mL and 5 µg / mL of puromycin screening solution, respectively. The solution was changed and the drug was added every 2-3 days, and the cell growth state was observed to determine that the maximum drug resistance pressure of the cell line was about 4 µg / mL; 4 µg / mL Puromycin was used for pressure screening to obtain a stable cell pool; (3) Monoclonal cell line screening After the cells were stabilized, monoclonal cells were screened by limiting dilution method with 3 µg / mL Puromycin. After plating on 96-well plates, the monoclonal cell line was determined by monoclonal imaging to obtain the recombinant cell 293-XB that can simultaneously express XBP1 and BIP. The monoclonal cell line with the best growth state was determined through step-by-step screening using 24-well plates and 6-well plates, and was frozen in time for simultaneous result verification.
6. An application of recombinant cell 293-XB for increasing antibody expression, characterized in that: The recombinant cell 293-XB is obtained by the method according to any one of claims 1 to 5, and the application comprises the following steps: (1) Select the target antibody and construct the PPT5 transient expression vector; (2) Use a plasmid extraction kit to extract plasmid DNA and purify the plasmid DNA; (3) 293-XB cells were revived using OPM-293 CD05 Medium. One day before antibody transfection, cells were cultured at 2×10 6 The cells were inoculated into fresh culture medium at a density of 3 × 10 cells / mL and cultured in a shaker at 37°C, 5% CO2, and 150 rpm. On the day of transfection, samples were taken to count the cell density and viability and the cell density was adjusted to 3 × 10 6 cells / mL, viability ≥95%; (4) The recombinant plasmid was transfected into the 293-XB cell line using PEI transfection reagent for expression. The specific transfection and expression process was as follows: plasmid DNA and PEI transfection reagent were diluted with OPM-293 CD05 Medium, and the diluted PEI transfection reagent was added to the diluted plasmid DNA at a ratio of plasmid DNA: PEI transfection reagent = 1:4; the complex was incubated at room temperature for about 20 minutes; 22 hours after transfection, 5% (v / v) 293-ProFeed was added to the shake flask. During the entire transient expression process, the glucose concentration was maintained above 4 g / L, and the cells were harvested when the cell viability was less than 70%; (5) The harvested target antibody was filtered through a 0.45 µm filter membrane and purified using a chromatography column filled with Protein A (GE) filler. The dissociation peak was collected and the protein content was detected using a spectrophotometer NanoDrop One and the protein purity was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE.