Application of Arnt2 gene in improving expression of exogenous recombinant protein of mammalian cells
By overexpressing the Arnt2 gene in CHO cells, constructing a recombinant vector, and transfecting the cells, the problem of low and unstable recombinant protein expression levels in CHO cells was solved, and the expression level and stability of recombinant proteins were significantly improved.
Patent Information
- Application Number
- CN202511258905.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-04
- Publication Date
- 2025-11-21
AI Technical Summary
The expression level of recombinant proteins in CHO cells is low and unstable, and existing technical modification methods have limited effectiveness.
By overexpressing the Arnt2 gene in CHO cells, a recombinant vector was constructed and transfected into the cells to form recombinant cells that stably overexpress Arnt2, which were then applied to a recombinant protein expression system.
It significantly improved the expression level of recombinant proteins and enhanced the stability of expression. For example, the expression level of recombinant adalimumab increased by 1.76-fold, stable expression increased by 2.85-fold, and the expression level was retained by 85.3% during passage.
Smart Images
Figure CN120989157A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to Arnt2 Application of a gene in improving expression of exogenous recombinant proteins in mammalian cells. BACKGROUND
[0002] Chinese hamsters ovary (CHO) cells are currently the preferred host cells for recombinant protein production and are widely used in the field of biopharmaceuticals. However, there are still problems such as low expression level and instability of expression of target proteins in the recombinant protein production system based on CHO cells.
[0003] Modifying cell lines by knocking out or overexpressing specific genes related to protein synthesis, secretion, cell cycle and apoptosis through biomolecular methods is a common method to improve the expression level of recombinant proteins in host cells. For example, the prior art reports that overexpression of FABP5 increased the yield of Herceptin monoclonal antibody by 20%; and knockdown of Aars1 increased the expression level of recombinant Adalimumab by 3.1 times.
[0004] Aryl Hydrocarbon Receptor Nuclear Translocator 2 (Arnt2) gene encodes a protein that is a transcription factor, which can form heterodimers with other proteins, bind to specific DNA sequences, and thus regulate gene transcription. Under hypoxic conditions, Arnt2 forms a complex with Aryl Hydrocarbon Receptor (AhR) and Hypoxia Inducible Factor 1α (HIF-1α), affecting various signal transduction pathways. Arnt2 It is expressed in various tumor tissues, and its expression level is related to cell proliferation and prognosis. In the field of cell engineering, Arnt2 its effect on the expression of recombinant proteins in cells needs to be studied. Arnt2 SUMMARY
[0005] In order to solve the problems of low expression level and instability of expression of recombinant proteins in CHO cells, the present application provides Arnt2 Application of a gene in improving expression of exogenous recombinant proteins in mammalian cells. The present application finds that Arnt2 the gene can regulate the expression of exogenous recombinant proteins in mammalian cells, and overexpression Arnt2 of the recombinant vector of the gene, the recombinant cell, and the recombinant protein expression system can improve the expression level and stability of recombinant proteins.
[0006] To achieve the above-mentioned purposes, the specific technical solutions of the present application are as follows: The present application provides a recombinant vector of a gene in the first aspect of the present applicationArnt2 The application of genes in enhancing the expression of exogenous recombinant proteins in mammalian cells, the aforementioned Arnt2 The nucleotide sequence of the gene is shown in SEQ ID No. 1.
[0007] Furthermore, the mammalian cell is a CHO cell.
[0008] Furthermore, overexpression in CHO cells Arnt2 Genes were used to enhance the expression level and stability of exogenous recombinant proteins in CHO cells.
[0009] Furthermore, the CHO cells are CHO-S, CHO-K1, CHO-DXB11, or CHO-DG44.
[0010] A second aspect of the present invention provides an overexpression Arnt2 A gene recombination vector, wherein the recombination vector is generated by the above-described... Arnt2 The gene was constructed after insertion into a starting vector, which is the piggyBac transposon.
[0011] Furthermore, the aforementioned Arnt2 The gene is inserted downstream of the CMV promoter in the starting vector.
[0012] A third aspect of the present invention provides a recombinant cell, wherein the recombinant cell is an overexpressing Arnt2 Recombinant cells of genes.
[0013] Furthermore, the recombinant cells are obtained by transfecting CHO cells with the recombinant vector described above.
[0014] Furthermore, the CHO cells are CHO-S, CHO-K1, CHO-DXB11, or CHO-DG44.
[0015] A fourth aspect of the present invention provides the application of the above-described recombinant vector or recombinant cell in enhancing the expression of exogenous recombinant proteins.
[0016] The fifth aspect of the present invention provides a recombinant protein expression system, which comprises the recombinant cells described above and an expression vector containing the gene encoding the target recombinant protein.
[0017] Furthermore, the target recombinant protein is a recombinant monoclonal antibody or a cytokine.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides Arnt2 Application of genes in enhancing the expression of exogenous recombinant proteins in mammalian cells. This invention discovers nucleotide sequences as shown in SEQ ID No. 1.Arnt2 Genes can regulate the expression of recombinant proteins in mammalian cells, so overexpression Arnt2 of genes in recombinant vectors can significantly improve the expression level of recombinant proteins. Experiments show that by transfecting recombinant vectors overexpressing Arnt2 into Chinese hamster ovary (CHO) cells, a stable CHO cell line overexpressing Figure 1 is obtained. When this cell line is used to express recombinant proteins, not only can the expression level of recombinant proteins be significantly improved, but also the stability of recombinant protein expression can be enhanced. BRIEF DESCRIPTION OF DRAWINGS
[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.
[0020] Arnt2 The recombinant vector for overexpression Figure 2 .
[0021] Arnt2 The recombinant vector for overexpression Arnt2 . Figure 3 The relative expression amount of mRNA in the recombinant cell (OE Arnt2) overexpressing
[0022] Arnt2 The transient expression level of recombinant adalimumab in the recombinant cell (OE Arnt2) overexpressing Figure 4 .
[0023] Arnt2 The stable expression level of recombinant adalimumab in the recombinant cell (OE Arnt2) overexpressing Figure 5 .
[0024] Arnt2 The maintenance rate of recombinant adalimumab expression after subculture of the recombinant cell (OE Arnt2) overexpressing FABP5 . DETAILED DESCRIPTION
[0025] The specific embodiments of the present application are described in detail below, but the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are all conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained commercially unless otherwise specified.
[0026] Chinese hamster ovary (CHO) cells are the preferred host for the production of recombinant proteins in biopharmaceuticals, but there are problems of low expression levels and instability. Current reports have shown that the production of recombinant proteins in CHO cells can be improved by genetic engineering, such as overexpression Aars1 or knockdown Arnt2 . Arnt2 is a transcription factor whose expression in tumor tissue is associated with cell proliferation. However, Arnt2 the effect on the expression of recombinant proteins in CHO cells needs further study.
[0027] The present application first constructs a recombinant vector overexpressing Arnt2 , then transfecting the recombinant vector overexpressing Arnt2 into host cells expressing recombinant proteins to obtain recombinant cells stably overexpressing Arnt2 . When this recombinant cell is used for recombinant protein expression, not only does it significantly improve the yield of recombinant proteins, but it also enhances the stability of recombinant protein expression.
[0028] Example 1: Arnt2 Construction of overexpression vector Arnt2 The sequence is shown as SEQ ID NO. 1, and the sequence of the gene is synthesized by General Biosystems Co., Ltd. Arnt2
[0029] SEQ ID NO. 1:
[0030] The synthesized Figure 1 gene was inserted downstream of CMV promoter in piggyBac transposon vector to construct a recombinant vector over-expressing Arnt2 , and the structure of the recombinant vector is shown in the following figure. The vector construction was completed by General Biosystems (Anhui) Co., Ltd., and the correctness of the constructed vector was verified by enzyme digestion and sequencing. Arnt2
[0031] Example 2: Construction of CHO cells over-expressing Arnt2 gene 1. Cell culture CHOS cells were inoculated in DMEM / F12 complete medium containing 10 v / v% inactivated fetal bovine serum and cultured at 37℃ and 5% CO2. One day before transfection, 4.0×10 5 cells per well were inoculated in a 12-well cell culture plate, and the cells were transfected when the confluence reached 70%~80%.
[0032] 2. Cell transfection The experiment was divided into: over-expression group (transfected with the recombinant plasmid over-expressing Arnt2 constructed in Example 1), control group (transfected with empty plasmid without Arnt2 gene) and untransfected group (without transfection).
[0033] ① 100 μL DMEM / F12 base medium was added to a 1.5 mL centrifuge tube, and 1.6 μg transfection plasmid (obtained by mixing recombinant plasmid over-expressing Arnt2 and transposase plasmid at a mass / volume ratio of 9:1) was added, and the mixture was mixed thoroughly and incubated at room temperature for 5 min.
[0034] ② Another centrifuge tube was prepared, and 100 μL DMEM / F12 base medium and 4 μL Lip2000 transfection reagent were added, and the mixture was mixed thoroughly and incubated at room temperature for 5 min.
[0035] ③ The mixture in step ② was added dropwise to the vector plasmid tube in step ①, and the mixture was mixed thoroughly and incubated at room temperature for 20 min.
[0036] ④ The cells plated for transfection the day before were changed to 1 mL fresh DMEM / F12 medium.
[0037] ⑤ The mixture in step ③ was added to the cells, and the mixture was mixed and then placed in an incubator for further culture. Fresh complete medium was replaced 4-6 h after transfection.
[0038] 3. Stable cell line screening: 48 h after cell transfection, replace the complete medium with a final concentration of 15 μg / mL puromycin for pressure screening, replace the medium every 2-3 days, and continue to screen for 1-2 weeks after the untransfected cells die and the final concentration of puromycin in the medium is reduced to 7.5 μg / mL.
[0039] 4. Overexpression Arnt2 Gene CHO cell identification Collect the stable cell lines screened after transfection, and use RT-qPCR to detect Arnt2 expression.
[0040] Centrifuge the CHO cells at 1000 rpm for 5 min, extract the total RNA of each group of cells using the Trizol method, and then reverse transcribe the RNA to cDNA using the HiScript IV All-in-One Ultra RT SuperMix for qPCR kit (purchased from Nanjing Novogene Bioinformatics Technology Co., Ltd.).
[0041] Design and synthesize primer sets for detecting Arnt2 and primer sets for detecting the internal reference βactin, the primer set for detecting Arnt2 contains the upstream primer Arnt2-F (as shown in SEQ ID NO. 2) and the downstream primer Arnt2-R (as shown in SEQ ID NO. 3), and the primer set for detecting the internal reference βactin contains the upstream primer β-actin-F (as shown in SEQ ID NO. 4) and the downstream primer β-actin-R (as shown in SEQ ID NO. 5).
[0042] SEQ ID NO. 2: 5'-AGAGAACTTAACTCAGGCCGC-3'.
[0043] SEQ ID NO. 3: 5'-CCAAGGCCATTCCTGAACCT-3'.
[0044] SEQ ID NO. 4: 5'-AGAGCAAGAGAGGTATTC-3'.
[0045] SEQ ID NO. 5: 5'-CCAGATCTTCTCCATATCG-3'.
[0046] Use the cDNA as a template and use the primer set for detecting Figure 2 or the primer set for detecting the internal reference βactin to perform RT-qPCR reaction: Each 20.0 μL reaction system contains: 10.0 μL SYBR Green Mix, 2.0 μL cDNA, 1.0 μL upstream primer, 1.0 μL downstream primer, and 6.0 μL enzyme-free water.
[0047] The reaction conditions were: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, for 40 cycles.
[0048] The melting curve stages are 95℃ for 15s, 60℃ for 60s, and 95℃ for 15s.
[0049] After RT-qPCR reaction, according to 2- △△CT calculate Arnt2 Relative expression of mRNA.
[0050] result Arnt2 As shown, overexpression Arnt2 In the CHO recombinant cell line (OE Arnt2) Arnt2 The mRNA expression level was significantly increased, 4.29 times higher than that of the control group.
[0051] Example 3: Recombinant Protein Expression System This embodiment uses the overexpression obtained from the screening and verification in Example 2. Figure 4 The CHO recombinant cells (OE Arnt2) were used for recombinant protein expression, including the construction of a recombinant protein expression system. The specific construction process is as follows: 1. Obtaining recombinant plasmids expressing adalimumab monoclonal antibody The recombinant plasmid expressing adalimumab used in this invention was constructed in the laboratory of this invention in the early stage. For specific information on the recombinant plasmid expressing adalimumab, please refer to application number 202411913965X, which is blast fungicide resistant.
[0052] 2. Transient expression system for recombinant adalimumab antibody The recombinant plasmid expressing adalimumab obtained in step 1 was transfected with... Arnt2 Overexpressing CHO cells and control CHO cells were transfected for 48 h and then cultured in serum-free suspension medium (purchased from Henan Punoyi Biological Products Research Institute Co., Ltd.) at 5×10⁻⁶ ppm. 5 Cells were seeded at a density of [number] cells / mL into 6-well plates and cultured in serum-free suspension at 37°C, 5% CO2, and 120 r / min. Cell density and viability were recorded daily after trypan blue staining using a Countstar automated cell counter. When cell viability decreased to below 70% (approximately day 7 of suspension), the supernatant was collected by centrifugation, and transient expression of recombinant adalimumab was detected using Western blotting.
[0053] Results are shown in Arnt2 Figure 2. The CHO recombinant cells overexpressing Figure 4 increased the transient expression of recombinant adalimumab by 1.76 times.
[0054] 3. Stable expression system of recombinant adalimumab The adalimumab expression vectors obtained in step 1 were respectively transfected into CHO cells Arnt2 The overexpression CHO cells and control CHO cells were transfected, and 48 h later, DMEM / F12 complete medium containing 15 μg / mL of final concentration of blasticidin was added for pressure screening. During the screening process, the medium was replaced every 2 days until the untransfected cells completely died. Then, the final concentration of blasticidin in the medium was reduced to 10 μg / mL for continued pressure screening for 2 weeks to obtain stable expression cell pools. The stable expression cell strains were inoculated into 6-well plates at 5 x 10 5 / mL, and serum-free suspension culture was carried out at 37°C, 5% CO2, and 120 r / min. When the cell viability was reduced to 70% (about 7th day of suspension culture), the supernatant was collected by centrifugation, and Western Blotting was used to detect the stable expression of recombinant adalimumab.
[0055] Results are shown in Arnt2 Figure 3. The CHO recombinant cells overexpressing Arnt2 increased the stable expression of recombinant adalimumab by 2.85 times.
[0056] 4. Long-term stable expression of recombinant protein The stable cell strains screened in Example 2 were subcultured, and the cells were subcultured once every 2-3 days, for a total of 10 generations. In the control group, it was observed that the expression of adalimumab was significantly reduced with the increase of subculture times, while the CHO recombinant cells overexpressing Figure 5 maintained stable expression of adalimumab throughout the culture period. After 10 generations, the expression of recombinant adalimumab in the control group was 62.3% of the initial expression, while the overexpression group retained 85.3% of the initial expression, indicating that the CHO recombinant cells overexpressing Arnt2 enhanced the stability of recombinant adalimumab expression in CHO cells. ).
[0057] It is to be understood that every range of values disclosed herein is to be understood to encompass any and every sub-range of values within the range. Although the preferred embodiments of the invention have been described above, it will be appreciated that those skilled in the art, on consideration of this disclosure, will be able to devise additional embodiments that, although not explicitly described or shown herein, nonetheless fall within the scope of the present invention. Accordingly, the appended claims are intended to include within their scope all such alternatives, modifications and variations as fall within the scope of the present invention. Various features and aspects of the present invention will become apparent from the following examples, which are intended only to exemplify the invention. It should be understood, of course, that in the various examples of the present invention, the specific phrasing of the claims will depend on the exact nature of the claims sought.
[0058] Obviously, numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims and their equivalents, the present application can be practiced otherwise than as specifically described herein.
Claims
1. Arnt2 The use of a gene in improving the expression of an exogenous recombinant protein in a mammalian cell, characterized in that, The Arnt2 The nucleotide sequence of the gene is shown in SEQ ID No.
1.
2. Use according to claim 1, characterized in that, The mammalian cell is a CHO cell.
3. Use according to claim 2, characterized in that, Overexpression in CHO cells Arnt2 Genes to improve the expression level and expression stability of exogenous recombinant proteins in CHO cells.
4. Use according to claim 3, characterized in that, The CHO cell is CHO-S, CHO-K1, CHO-DXB11 or CHO-DG44.
5. A recombinant vector overexpressing Arnt2 a gene, characterized in that, The recombinant vector is constructed by inserting the gene of claim 1 into a starting vector, which is a piggyBac transposon. Arnt2 The recombinant vector is constructed by inserting the gene of claim 1 into a starting vector, which is a piggyBac transposon.
6. The recombinant vector of claim 5, wherein, The Arnt2 The gene was inserted into the downstream of the CMV promoter in the starting vector.
7. A recombinant cell, wherein, The recombinant cell is a CHO cell overexpressing Arnt2 A recombinant cell of the gene obtained after transfecting a CHO cell with the recombinant vector of claim 5.
8. Use of the recombinant vector of claim 5 or the recombinant cell of claim 7 in improving expression of an exogenous recombinant protein.
9. A recombinant protein expression system, characterized in that, The recombinant protein expression system consists of the recombinant cell of claim 7 and an expression vector containing a gene encoding the recombinant protein of interest.
10. The recombinant protein expression system of claim 9, wherein, The recombinant protein of interest is a monoclonal antibody or a cytokine.