Signal peptide combination for improving antibody expression quantity, recombinant expression vector, recombinant cell and application

Abstract

The invention provides a signal peptide combination for improving antibody expression quantity, a recombinant expression vector, a recombinant cell and application, and belongs to the technical field of bioengineering. According to the invention, three signal peptides SP-A, SP-B and SP-C with good expression are combined pairwise and fused with an Inflixi or human immune globulin antibody in Chinese hamster ovary cells for expression. Compared with the original carrier signal peptide, the combined signal peptide has the advantages that the antibody expression quantity is obviously improved, and the problem of low antibody expression quantity is solved. The antibody produced by the method is high in yield and convenient for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and particularly relates to a combination of signal peptides for improving antibody expression, a recombinant expression vector, recombinant cells, and applications. Background Technology

[0002] Antibody expression technology has reached a relatively mature stage and is widely used in scientific research, diagnosis, and treatment. In terms of expression systems, mammalian cell expression systems (such as CHO and HEK293 cells) have become the gold standard for therapeutic antibody production due to their ability to perform complex post-translational modifications (such as glycosylation). However, antibody expression still faces the problem of low expression levels. Examples include infliximab and some human immunoglobulin antibodies. Infliximab is a human-mouse chimeric monoclonal antibody targeting tumor necrosis factor-α (TNF-α), primarily used clinically to treat various autoimmune diseases such as rheumatoid arthritis, Crohn's disease, and ulcerative colitis. It was the world's first approved anti-TNF-α antibody drug for treating these diseases, and market demand is huge. However, Infliximab is a typical "difficult protein," with extremely low expression efficiency in conventional eukaryotic expression systems. Even with traditional strong promoters and optimized culture processes, the expression titer of Infliximab still falls short of the cost control requirements for industrial production.

[0003] Signal peptides, as key components guiding protein transmembrane transport, have a decisive impact on the correct folding and secretion efficiency of proteins. In current technologies, antibody expression typically utilizes the heavy and light chain signal peptides of natural antibodies, such as the heavy chain signal peptide of murine IgG or the light chain signal peptide of human IgG1. Studies have shown that these natural signal peptides have insufficient compatibility with the secretory system of CHO cells, exhibiting problems such as low signal peptide cleavage efficiency and protein aggregation due to retention in the endoplasmic reticulum, directly limiting the enhancement of antibody expression levels.

[0004] To address the issue of signal peptide efficiency, developing a dedicated optimized signal peptide tailored to the structural characteristics of antibodies is crucial for overcoming their expression bottleneck. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a signal peptide combination, recombinant expression vector, recombinant cells and applications for improving antibody expression levels, overcoming the problems of low antibody expression levels and insufficient efficiency of traditional signal peptides in the prior art. By directionally modifying the core functional regions of the signal peptides, the secretory expression efficiency of antibodies in CHO cells is significantly improved.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a combination of signal peptides to enhance antibody expression, comprising a heavy chain signal peptide and a light chain signal peptide, wherein the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0007] The present invention also provides a gene encoding the combination of signal peptides described above, wherein the nucleotide sequence encoding SEQ ID NO:1 is shown in SEQ ID NO:4, the nucleotide sequence encoding SEQ ID NO:2 is shown in SEQ ID NO:5, and the nucleotide sequence encoding SEQ ID NO:3 is shown in SEQ ID NO:6.

[0008] The present invention also provides a recombinant expression vector, comprising the aforementioned gene and the original expression vector.

[0009] Preferably, the original expression vector includes the pWTY240204 vector, the heavy chain gene is regulated by the CMV promoter, the light chain gene is regulated by the EF-1α promoter, and the pWTY240204 vector also contains the selection marker gene blastomycin.

[0010] The present invention also provides a recombinant cell comprising the recombinant expression vector and a host cell, wherein the host cell comprises Chinese hamster ovary cells.

[0011] The present invention also provides a method for increasing antibody expression levels, comprising the following steps: 1) The gene is cloned into the original expression vector to construct the recombinant expression vector; 2) The recombinant expression vector was transfected into Chinese hamster ovary cells to obtain the recombinant cells; 3) Screening to obtain recombinant cell lines that stably express high levels of antibodies; 4) Cultivate the recombinant cell line, collect and purify the expression product to obtain the corresponding antibody.

[0012] Preferably, the transfection method in step 2) is liposome transfection, and the screening in step 3) uses blast fungicide.

[0013] The present invention also provides the application of the aforementioned signal peptide combination, the aforementioned gene, the aforementioned recombinant expression vector, or the aforementioned recombinant cell in the preparation of antibodies.

[0014] Preferably, the antibody includes infliximab and human immunoglobulin.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention replaces the light chain and heavy chain signal peptide gene sequences of antibody vectors with optimized signal peptide sequences that combine SP-A, SP-B, and SP-C in pairs. After optimization, SP-A, SP-B, and SP-C are combined in pairs to replace the original signal peptides, resulting in expression vectors SP-1, SP-2, SP-3, SP-4, SP-5, SP-6, SP-7, SP-8, and SP-9. These vectors are then integrated into the genes of CHO cells, achieving stable and efficient expression of antibodies containing optimized signal peptides in CHO cells, significantly increasing the antibody expression level.

[0016] This invention replaces the original antibody signal peptide with an optimized signal peptide and applies it to construct a recombinant protein expression system. An expression vector containing the optimized signal peptide is transfected into CHO cells to construct a CHO cell line expressing the antibody gene containing the optimized signal peptide. This CHO cell line significantly improves antibody protein expression levels. Furthermore, as a host cell for recombinant protein expression, this CHO cell line, compared to wild-type CHO cell lines, enhances recombinant protein expression levels, effectively overcoming the problems of low protein levels and difficulty in expression present in current CHO cell expression systems. The optimized signal peptide is fully compatible with existing CHO cell expression systems, requiring no modification to production equipment or processes, and can be directly applied to industrial production, significantly reducing the production cost per unit antibody. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of a gene vector structure expressing an antibody containing an optimized signal peptide; Figure 2 This describes the expression level of Infliximab-SP antibody in the CHO cell line expressing the pWTY240204LH-Inf gene; Figure 3 This shows the expression level of Ig-SP antibody in the CHO cell line expressing the pWTY240204LH-Ig gene. Detailed Implementation

[0018] This invention provides a combination of signal peptides to enhance antibody expression, comprising a heavy chain signal peptide and a light chain signal peptide, wherein the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0019] In this invention, the amino acid sequence of SEQ ID NO:1 is specifically MEFGLSWVFLVALFRGVQC, the amino acid sequence of SEQ ID NO:2 is specifically MDMRVPAQLLGLLLLWLSGARC, and the amino acid sequence of SEQ ID NO:3 is specifically MKFLVNVALVFMVVYISYIYA.

[0020] The present invention also provides a gene encoding the combination of signal peptides described above, wherein the nucleotide sequence encoding SEQ ID NO:1 is shown in SEQ ID NO:4, the nucleotide sequence encoding SEQ ID NO:2 is shown in SEQ ID NO:5, and the nucleotide sequence encoding SEQ ID NO:3 is shown in SEQ ID NO:6.

[0021] In this invention, the nucleotide sequence of SEQ ID NO:4 is specifically ATGGAGTTCGGCCTGAGCTGGGTGTTTCTGGTGGCCCTGTTCAGGGGCGTGCAGTGT, the nucleotide sequence of SEQ ID NO:5 is specifically ATGGATATGCGGGTGCCTGCTCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGCGGCGCTAGGTGC, and the nucleotide sequence of SEQ ID NO:6 is specifically ATGAAGTTCCTGGTGAACGTGGCCCTGGTGTTCATGGTGGTGTACATCTCCTACATCTACGCT.

[0022] The present invention also provides a recombinant expression vector, comprising the aforementioned gene and the original expression vector.

[0023] In this invention, the original expression vector includes the pWTY240204 vector, the heavy chain gene is regulated by the CMV promoter, the light chain gene is regulated by the EF-1α promoter, and the pWTY240204 vector also contains the selection marker gene blast fungicide.

[0024] This invention also provides a recombinant cell, comprising the recombinant expression vector and a host cell, wherein the host cell comprises Chinese hamster ovary cells. In this invention, the Chinese hamster ovary cells are preferably CHO-S cells.

[0025] The present invention also provides a method for increasing antibody expression levels, comprising the following steps: 1) The gene is cloned into the original expression vector to construct the recombinant expression vector; 2) The recombinant expression vector was transfected into Chinese hamster ovary cells to obtain the recombinant cells; 3) Screening to obtain recombinant cell lines that stably express high levels of antibodies; 4) Cultivate the recombinant cell line, collect and purify the expression product to obtain the corresponding antibody.

[0026] In this invention, the transfection method in step 2) is liposome transfection, and the screening in step 3) uses blast fungicide.

[0027] This invention also provides the use of the aforementioned signal peptide combination, the aforementioned gene, the aforementioned recombinant expression vector, or the aforementioned recombinant cell in the preparation of antibodies. In this invention, the antibody comprises infliximab and human immunoglobulin.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] 1. Construct an expression vector for Infliximab antibody.

[0031] Figure 1 As shown. The optimized signal peptide gene sequences, combining SP-A, SP-B, and SP-C signal peptides in pairs, replace the light chain signal peptide gene sequence ATGAAGTGGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGGTGACCGGC (SEQ ID NO: 9) and the heavy chain signal peptide gene sequence ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGAGAG (SEQ ID NO: 10) of the Infliximab vector. The specific nucleotide sequences of SP-A, SP-B, and SP-C are as follows: SP-A: ATGGAGTTCGGCCTGAGCTGGGTGTTTCTGGTGGCCCTGTTCAGGGGCGTGCAGTGT (SEQ ID NO: 4), SP-B: ATGGATATGCGGGTGCCTGCTCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGCGGCGCTAGGTGC (SEQ ID NO: 4), SP-B: ATGGATATGCGGGTGCCTGCTCAGCTGCTGGGCCTGCTGTGGCTGCGGCGCTAGGTGC (SEQ ID NO: 9). .5), SP-C: ATGAAGTTCCTGGTGAACGTGGCCCTGGTGTTCATGGTGGTGTACATCTCCTACATCTACGCT (SEQ ID NO.6).

[0032] 2. Construct an expression vector for human immunoglobulin 294 antibody.

[0033] Figure 1 As shown. The optimized signal peptide gene sequences, combining SP-A, SP-B, and SP-C signal peptides in pairs, replace the light chain signal peptide gene sequence ATGAAGTGGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGGTGACCGGC (SEQ ID NO: 9) and the heavy chain signal peptide gene sequence ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGAGAG (SEQ ID NO: 10) of the human immunoglobulin 294 antibody vector. The specific nucleotide sequences of SP-A, SP-B, and SP-C are as follows: SP-A: ATGGAGTTCGGCCTGAGCTGGGTGTTTCTGGTGGCCCTGTTCAGGGGCGTGCAGTGT (SEQ ID NO: 4), SP-B: ATGGATATGCGGGTGCCTGCTCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGCGGCGCTAGGTGC (SEQ ID NO: 4), SP-B: ATGGATATGCGGGTGCCTGCTCAGCTGCTGGGCCTGCTGTGGCTGCGGCGCTAGGTGC (SEQ ID NO: 4). .5), SP-C: ATGAAGTTCCTGGTGAACGTGGCCCTGGTGTTCATGGTGGTGTACATCTCCTACATCTACGCT (SEQ ID NO.6).

[0034] The original light and heavy chains of the signal peptides in plasmids pWTY240204LH-Inf and pWTY240204LH-Ig were replaced with nine optimized signal peptide combinations. The optimized signal peptide sequences are shown in Table 1 (synthesized by General Biotech (Anhui) Co., Ltd.). The optimized SP plasmids of the corresponding antibodies were obtained and named as follows: Antibody-SP-1, Antibody-SP-2, Antibody-SP-3, Antibody-SP-4, Antibody-SP-5, Antibody-SP-6, Antibody-SP-7, Antibody-SP-8, and Antibody-SP-9.

[0035] Table 1 Optimized signal peptide combinations

[0036] Example 2

[0037] Constructing CHO cell lines expressing their respective antibodies and the expression of the corresponding antibodies.

[0038] CHO-S cells (purchased from Gibco) were cultured in DMEM / F12 medium containing 10% inactivated fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase, at a density of 2.0 × 10⁶ cells per well. 5A certain number of cells were seeded into 12-well cell culture plates and seeded for 24 hours. When the cell confluence reached about 85%, the cells were used for cell transfection.

[0039] Add 50 μL to 0.5 mL of serum-free culture medium (purchased from Henan Punoyi Biological Products Research Institute Co., Ltd.) In DMEM / F12 medium, the optimized pWTY240204LH-Inf vectors Infliximab-SP-1, Infliximab-SP-2, Infliximab-SP-3, Infliximab-SP-4, Infliximab-SP-5, Infliximab-SP-6, Infliximab-SP-7, Infliximab-SP-8, Infliximab-SP-9 constructed in Example 1 and the pWTY240204LH-Ig vectors Ig-SP-1, Ig-SP-2, Ig-SP-3, Ig-SP-4, Ig-SP-5, Ig-SP-6, Ig-SP-7, Ig-SP-8, Ig-SP-9 were transfected into CHO-S cells using liposome transfection to construct the Infliximab-SP CHO / Ig-SP CHO cell lines were used as the overexpression (Infliximab-SP / Ig-SP) group. Meanwhile, the original CHO cell lines expressing pWTY240204LH-Inf / pWTY240204LH-Ig served as the control group.

[0040] The liposome transfection method is as follows: (1) Taking a 24-well plate as an example, take a 1.5 mL centrifuge tube and label it with the name of the vector. Add 50 μL of basic DMEM / F12 medium to the tube, and then add 0.8 μg of the vector expressing Infliximab-SP / Ig-SP constructed in Example 1 to the tube. Mix well and let stand at room temperature for 5 min. (2) Take another centrifuge tube, label it as the transfection reagent tube, add 50 μL of basic DMEM / F12 medium into the tube, add 2 μL of transfection reagent Lipofectamine 3000 into the tube, mix thoroughly, and let stand at room temperature for 5 min. (3) Add the mixture from step (2) to the plasmid tube from step (1), mix thoroughly, and let stand at room temperature for 20 minutes; (4) Remove the cells that were plated the day before for transfection from the incubator, discard the old culture medium, wash each well twice with 1×PBS buffer, and then add 500 μL of fresh DMEM / F12 serum-depleted culture medium without antibiotics. (5) Add the mixture from step (3) to the wells, gently shake the culture plate to distribute the mixture evenly, and then place it in an incubator for culture. After 5 hours of incubation, remove the culture plate, replace it with fresh complete culture medium, and continue culturing. After 48 hours of cell transfection, use the above-mentioned optimal BSD concentration (15 μg / mL) for cell selection. Once the cells stop dying after drug treatment, count the cell suspension and seed one cell per well into a 96-well plate, waiting for cell proliferation. When the cell confluence rate in the 96-well plate reaches about 90%, expand the culture, and then proceed with the subsequent passage and selection steps.

[0041] Forty-eight hours after transfection, recombinant CHO cells were cultured in six-well plates in 3 mL of protein-free, serum-free, and chemically defined CD CHO medium (Henan Punoyi Biological Products Research Institute Co., Ltd.) for 6 days until the cell count reached 1×10⁶. 7 Cells were collected daily and their density and viability were measured using a Countstar BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.). On day 7, the supernatant was collected by centrifugation and quantified using the same cell count for analysis of recombinant Infliximab monoclonal antibody / Ig antibody expression. Western blot analysis was used to detect antibody expression in CHO cells of the SP and Control groups.

[0042] Experimental results: such as Figure 2 As shown, the results indicate that the optimized signal peptide combination can significantly improve the expression level of recombinant infliximab, with Infliximab-SP-7 expression increasing by 3.4-fold and Infliximab-SP-8 expression increasing by 4.7-fold.

[0043] like Figure 3 As shown, the results indicate that the optimized signal peptide combination can significantly improve the expression level of recombinant human immunoglobulin antibodies, with Ig-SP-7 expression increasing by 5.6 times and Ig-SP-8 expression increasing by 4.8 times.

[0044] As shown in the above embodiments, this invention expresses three well-expressed signal peptides, SP-A, SP-B, and SP-C, in pairs by fusing them with an antibody in Chinese hamster ovary cells. Compared with the original vector signal peptides, the combined signal peptides of this invention significantly increase antibody expression levels, solving the problem of low antibody expression. The method of this invention produces high-yield antibodies, facilitating industrial application.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination of signal peptides to enhance antibody expression, characterized in that, It includes a heavy chain signal peptide and a light chain signal peptide, wherein the amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and the amino acid sequence of the light chain signal peptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

2. A gene encoding the signal peptide combination of claim 1, characterized in that, The nucleotide sequence encoding SEQ ID NO:1 is shown in SEQ ID NO:4, the nucleotide sequence encoding SEQ ID NO:2 is shown in SEQ ID NO:5, and the nucleotide sequence encoding SEQ ID NO:3 is shown in SEQ ID NO:

6.

3. A recombinant expression vector, characterized in that, Includes the gene and original expression vector as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The original expression vector includes the pWTY240204 vector, in which the heavy chain gene is regulated by the CMV promoter and the light chain gene is regulated by the EF-1α promoter. The pWTY240204 vector also contains the selection marker gene blast fungicide.

5. A recombinant cell, characterized in that, It includes the recombinant expression vector and host cell as described in claim 3 or 4, wherein the host cell includes Chinese hamster ovary cells.

6. A method for increasing antibody expression levels, characterized in that, Includes the following steps: 1) The gene described in claim 2 is cloned into the original expression vector to construct the recombinant expression vector described in claim 3 or 4; 2) Transfect the recombinant expression vector of claim 3 or 4 into Chinese hamster ovary cells to obtain the recombinant cells of claim 5; 3) Screening to obtain recombinant cell lines that stably express high levels of antibodies; 4) Cultivate the recombinant cell line, collect and purify the expression product to obtain the corresponding antibody.

7. The method according to claim 6, characterized in that, Step 2) The transfection method is liposome transfection, and Step 3) The screening uses blast fungicide.

8. The use of the signal peptide combination of claim 1, the gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 5 in the preparation of antibodies.

9. The application according to claim 8, characterized in that, The antibodies include infliximab and human immunoglobulin.

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