Method for improving expression level of recombinant protein, gene engineering cell, kit and expression system

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a method for improving the expression level of recombinant protein, a gene engineering cell, a kit and an expression system. The miR-21-5p is screened and verified to be capable of serving as a genetic engineering modification target of mammalian cells (specifically CHO cells), a miR-21-5p overexpressed recombinant protein stable expression system is constructed, and the expression quantity of the recombinant protein can be increased; furthermore, the miR-21-5p and the miR-92a-3p are creatively combined to serve as a gene engineering modification target, and compared with an expression system constructed by a single target, the expression quantity of the recombinant protein can be further improved by constructing a recombinant protein stable expression system for miR-21-5p overexpression and miR-92a-3p expression inhibition.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a method for improving the expression level of a recombinant protein, a genetic engineering cell, a kit and an expression system. Background Art

[0002] In recent years, the demand for recombinant protein drugs in the pharmaceutical market has continued to grow. Among them, recombinant antibody drugs retain the specificity and biological activity of natural antibodies while eliminating the immunogenicity of antibodies. They have been used in the diagnosis and treatment of many diseases. Since mammalian cells have the functions of protein folding and post-translational modification, the properties of expressed proteins are close to those of natural proteins, and they have become an important platform for the production of recombinant protein drugs. Chinese hamster ovary (CHO) cells have a post-translational modification system similar to that of human cells, are easy to acclimate in suspension, and can be cultured without serum and protein. They have become the main platform for the current research and development and production of biopharmaceuticals. CHO cells have been successfully used to produce many valuable biotechnology drugs such as cytokines, recombinant enzymes, recombinant antibodies, and recombinant fusion proteins, making a significant contribution to market demand. At present, the expression level of recombinant antibodies has been improved to a certain extent through methods such as expression vector optimization, cell line transformation, and culture process improvement, but the yield and activity of recombinant antibody drugs produced using CHO cells still cannot fully meet market demand.

[0003] MicroRNA (miRNA) is a non-coding single-stranded RNA derived from endogenous chromosomes that can regulate biological processes such as cell proliferation, differentiation, immune regulation, and metabolism. Since these endogenous small RNAs can regulate all cellular pathways, miRNAs are widely used in CHO cell engineering to improve the expression of target proteins. A single miRNA can bind to multiple target genes without increasing the translation burden of the cell to regulate different metabolic pathways, and when regulating different metabolic pathways that are beneficial to antibody production, an additive effect may occur. With the continuous development of high-throughput omics technology, multi-omics analysis of CHO cells has provided new strategies for cell line transformation and productivity improvement, and miRNA can be used as a potential target for cell engineering. Although some studies have suggested that some miRNA targets affect the expression performance of recombinant proteins in CHO cell lines, the strategies that can be applied in industry are relatively limited overall. Therefore, it is necessary to carry out continuous innovative research on miRNA targets to provide new strategies for improving the expression performance and yield of recombinant proteins. Summary of the invention

[0004] In order to overcome the defects of the prior art, one of the objects of the present invention is to provide a method for improving the expression level of recombinant protein, wherein miR-21-5p is used as a genetic engineering target, or combined with miR-92a-3p as a genetic engineering target, and the expression level of recombinant protein in mammalian cells is improved by increasing the expression of miR-21-5p, or increasing the expression of miR-21-5p and simultaneously inhibiting the expression of miR-92a-3p.

[0005] The second object of the present invention is to provide a genetically engineered cell, in which miR-21-5p is overexpressed and miR-92a-3p expression is inhibited, and as a host cell, it is capable of efficiently expressing and producing recombinant proteins.

[0006] The third object of the present invention is to provide a kit for constructing a recombinant protein expression system, using the cell line with overexpression of miR-21-5p and inhibition of miR-92a-3p expression provided by the present invention as a host cell to achieve efficient production of recombinant protein.

[0007] The fourth object of the present invention is to provide a recombinant protein expression system, which is constructed using the cell line with overexpression of miR-21-5p and inhibition of miR-92a-3p expression provided by the present invention as a host cell to achieve efficient production of recombinant proteins.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0009] A method for increasing the expression of a recombinant protein, and increasing the expression of miR-21-5p in a recombinant protein expression system;

[0010] Alternatively, the expression level of miR-21-5p in the recombinant protein expression system is increased, and the expression level of miR-92a-3p is decreased.

[0011] In a specific embodiment of the present invention, the recombinant protein expression system is a CHO cell capable of expressing recombinant protein;

[0012] Optionally, a recombinant protein expression system for overexpression of miR-21-5p and inhibition of miR-92a-3p expression is constructed by co-transfecting miR-21-5p mimics, or miR-21-5p mimics and miR-92a-3 inhibitor into a recombinant protein expression system.

[0013] In a specific embodiment of the present invention, miR-21-5p mimics and miR-92a-3p inhibitor are co-transfected into CHO cells expressing recombinant proteins to construct CHO cells expressing recombinant proteins in which miR-21-5p is overexpressed and miR-92a-3p expression is inhibited.

[0014] Optionally, the nucleotide sequence of miR-21-5p mimics is shown in SEQ ID NO.1; the nucleotide sequence of miR-92a-3pinhibitor is shown in SEQ ID NO.2.

[0015] In a specific embodiment of the present invention, the final transfection concentration of the miR-21-5p mimics, or miR-21-5p mimics and miR-92a-3p inhibitor is 30 nM.

[0016] Optionally, the transfection dosage ratio of the miR-21-5p mimics and the miR-92a-3p inhibitor is 1:1.

[0017] A genetically engineered cell, wherein the cell is used as a host cell for recombinant protein expression, wherein miR-21-5p is overexpressed; or miR-21-5p is overexpressed and miR-92a-3p expression is suppressed. Optionally, the cell is a CHO cell.

[0018] A kit, characterized in that it comprises the above-mentioned genetically engineered cells, a recombinant protein expression vector containing a target protein encoding gene, and a transfection reagent;

[0019] Or include CHO cells, miR-21-5p mimics, miR-92a-3p inhibitor, recombinant protein expression vector, and transfection reagent.

[0020] The expression system was constructed using the above kit.

[0021] In a specific embodiment of the present invention, the recombinant protein is adalimumab. It should be understood by those skilled in the art that the scheme of the present invention can be applied to the expression of other recombinant proteins, not limited to adalimumab.

[0022] The above-mentioned recombinant protein expression system can be used to prepare a preparation containing the target protein; the preparation is selected from a protein detection reagent, a target protein drug for treating or preventing a disease, and a gene drug carrying the target protein.

[0023] Beneficial effects of the present invention:

[0024] The present invention aims to provide a new strategy for improving the production capacity of recombinant proteins. It screens and verifies that miR-21-5p can be used as a genetic engineering target for mammalian cells (specifically CHO cells). By constructing miR-21-5p overexpressing cells as host cells for recombinant protein expression, a recombinant protein stable expression system is constructed to increase the expression amount of the recombinant protein. The specific embodiments of the present invention verify that it can be applied to increase the stable expression amount of the recombinant adalimumab antibody.

[0025] Furthermore, the present invention creatively combines miR-21-5p and miR-92a-3p as genetic engineering targets for mammalian cells (specifically CHO cells), and constructs cells with overexpression of miR-21-5p and inhibition of miR-92a-3p expression as host cells for recombinant protein expression to construct a stable recombinant protein expression system. Compared with the expression system constructed with a single target, the expression amount of the recombinant protein can be further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of miR-21-5p on adalimumab expression was detected by Western blot in Example 1;

[0027] Figure 2 This is the Western blot detection in Example 2 to examine the effect of combined miR-21-5p and miR-92a-3p on adalimumab expression. DETAILED DESCRIPTION

[0028] Unless otherwise specified, the relevant operations in the embodiments and test examples are all conventional technical means in the field; the various culture media, reagents, etc. used in the embodiments and test examples are all commercially available products.

[0029] The cells used in the following examples are recombinant adalimumab monoclonal cell lines constructed and screened in the laboratory in the early stage. The specific method is: the independently constructed adalimumab plasmid is transfected into CHO cells, and the cell pool is obtained by screening with blasticidin for 2 to 3 weeks, and the recombinant adalimumab monoclonal cell line is obtained by screening with limiting dilution method.

[0030] The miR-21-5p mimics and mimics NC, miR-21-5p inhibitor, miR-92a-3pin inhibitor and inhibitor NC used were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0031] The following examples are only used to further illustrate the present invention, but do not constitute any limitation to the present invention.

[0032] Example 1 Detection of the effect of miR-21-5p on adalimumab expression

[0033] (1) Cell recovery

[0034] Resuscitate the recombinant adalimumab monoclonal cell line stored in liquid nitrogen, put it in a 37°C water bath for rapid thawing, add DMEM / F12 complete medium to the cells in an ultra-clean workbench, mix by pipetting, and transfer to a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5min, collect the cells, add 1mL DMEM / F12 complete medium, mix by pipetting, and transfer to a cell culture flask, add 2mL complete medium, and culture in a 37°C, 5% CO2 cell culture incubator.

[0035] (2) Cell transfection

[0036] The cell morphology and growth status were observed under an inverted microscope. When the cells covered 80-90% of the bottom area of ​​the culture flask, the cells were digested, collected and counted. 2×10 5 Inoculate cells into 12-well cell culture plates, add 1 mL of DMEM / F12 complete medium to each well, and culture in a 37°C, 5% CO2 incubator. When the cell growth density reaches 70-90% confluence, use Lipofectamine TM 2000 transfection reagent was used to transfect 30 nM miR-21-5p mimics and mimics NC, miR-21-5p inhibitor and inhibitor NC, respectively. After culturing in a cell culture incubator for 4-6 h, the culture medium was replaced with DMEM / F12 complete medium for continued culture.

[0037] (3) Cell suspension

[0038] 48 h after transfection, cells were digested with trypsin to form a single cell suspension, and 1 mL of CHO cell serum-free medium (purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd.) was added to resuspend the cells. The density of live cells was detected using a Countstar Rigel S2 cell counter. 5 Cells were inoculated into a six-well cell culture plate at a cell density of 10 cells / mL, 3 mL of CHO cell serum-free medium was added to each well, and the culture plate was placed on a shaker in a 37°C, 5% CO2 cell culture incubator, and the rotation speed was adjusted to 120 rpm for suspension culture.

[0039] (4) Western blot detection of adalimumab expression level

[0040] After 7 days of cell suspension culture, the cell suspension was collected and centrifuged at 1000 rpm for 5 min. The supernatant was collected and boiled for 10 min with 5× protein loading buffer. The expression of adalimumab was detected by Western blot. Figure 1As shown, the expression of adalimumab in the miR-21-5p mimics transfection group was significantly increased, and compared with the control group mimics NC, the expression of adalimumab increased by 1.38 times; while the expression of adalimumab in the miR-21-5p inhibitor transfection group was significantly decreased, and compared with the control group inhibitor NC, the expression of adalimumab decreased by 0.59 times.

[0041] Example 2 Detection of the effect of combined miR-21-5p and miR-92a-3p on adalimumab expression

[0042] (1) Cell transfection

[0043] According to 2×10 per well 5 Inoculate cells into 12-well cell culture plates, add 1 mL of DMEM / F12 complete medium to each well, and culture in a 37°C, 5% CO2 incubator. When the cell growth density reaches 70-90% confluence, use Lipofectamine TM 30 nM miR-21-5p mimics, 30 nM miR-92a-3p inhibitor and 30 nM miR-21-5p mimics+30 nM miR-92a-3p inhibitor were transfected with 2000 transfection reagent, and after culturing in a cell culture incubator for 4-6 h, the culture medium was replaced with DMEM / F12 complete medium for continued culture.

[0044] (2) Western blot detection of the effect of combined miR-21-5p and miR-92a-3p on adalimumab expression

[0045] According to the cell suspension culture method in Example 1, 5×10 5 The control group cells, cells transfected with miR-21-5p mimics, miR-92a-3p inhibitor, and miR-21-5p mimics+miR-92a-3 inhibitor (miR-21-5p mimics: miR-92a-3p inhibitor=1:1) were inoculated into six-well cell culture plates at a cell density of cells / mL, and 3 mL of CHO cell serum-free medium was added to each well. The culture plates were placed in a 37°C, 5% CO2 cell culture incubator with a shaker at 120 rpm for suspension culture. After 7 days of suspension culture, the cell suspension was collected, the supernatant was collected after centrifugation, and the expression level of adalimumab was detected by Western blot. The results are shown in Figure 2As shown in the figure, compared with the control group, transfection of miR-21-5p mimics and miR-92a-3 inhibitor increased the expression of adalimumab by 1.38 times and 1.91 times, respectively. Compared with transfection of miR-21-5p mimics or miR-92a-3p inhibitor alone, co-transfection of miR-21-5p mimics + miR-92a-3 inhibitor further increased the expression of adalimumab by 2.30 times.

[0046] The verification results of the above examples show that simultaneously overexpressing miR-21-5p and inhibiting miR-92a-3p can significantly increase the expression level of recombinant adalimumab in CHO cells, providing a new strategy for increasing the expression level of recombinant proteins.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for increasing the expression of a recombinant protein, characterized in that: Increase the expression of miR-21-5p in the recombinant protein expression system; Alternatively, the expression level of miR-21-5p in the recombinant protein expression system is increased, and the expression level of miR-92a-3p is decreased.

2. The method for increasing the expression of a recombinant protein according to claim 1, wherein: The recombinant protein expression system is a CHO cell capable of expressing recombinant protein.

3. The method for increasing the expression of a recombinant protein according to claim 2, wherein: A recombinant protein expression system for overexpression of miR-21-5p and inhibition of miR-92a-3p expression was constructed by co-transfecting miR-21-5p mimics, or miR-21-5p mimics and miR-92a-3p inhibitor into the recombinant protein expression system.

4. The method for increasing the expression level of a recombinant protein according to claim 3, characterized in that: The nucleotide sequence of miR-21-5p mimics is shown in SEQ ID NO.1; the nucleotide sequence of miR-92a-3p inhibitor is shown in SEQ ID NO.

2.

5. The method for increasing the expression level of a recombinant protein according to claim 4, characterized in that: The final transfection concentration of the miR-21-5pmimics, or miR-21-5p mimics and miR-92a-3p inhibitor is 30 nM.

6. The method for increasing the expression level of a recombinant protein according to claim 5, characterized in that: The transfection dosage ratio of the miR-21-5pmimics and miR-92a-3p inhibitor is 1:

1.

7. A genetically engineered cell, characterized in that The cell is used as a host cell for recombinant protein expression, wherein miR-21-5p is overexpressed; or miR-21-5p is overexpressed and miR-92a-3p expression is suppressed.

8. The genetically engineered cell according to claim 7, characterized in that The cells are CHO cells. Optionally, the genetically engineered cells are constructed by co-transfecting miR-21-5p mimics, or miR-21-5p mimics and miR-92a-3p inhibitor into a CHO cell line; Optionally, the genetically engineered cell is constructed by co-transfecting a miR-21-5p overexpression vector and / or a miR-92a-3p inhibition expression vector into a CHO cell line; Optionally, the nucleotide sequence of miR-21-5p mimics is as shown in SEQ ID NO.1; the nucleotide sequence of miR-92a-3pinhibitor is as shown in SEQ ID NO.

2.

9. A kit, characterized in that Comprising the genetically engineered cell according to claim 7 or 8, a recombinant protein expression vector containing a target protein encoding gene, and a transfection reagent; Or include CHO cells, miR-21-5p mimics, miR-92a-3p inhibitor, recombinant protein expression vector, and transfection reagent.

10. An expression system, characterized in that The kit is constructed according to claim 9. Optionally, the recombinant protein is adalimumab.

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