Use of let-7a in the expression of recombinant proteins in CHO cells and expression systems

Abstract

The present invention relates to the field of genetic engineering, specifically to the use of let-7a in recombinant protein expression in CHO cells and an expression system. Increasing the amount of miRNA let-7a in a CHO cell recombinant protein expression system increases the expression level of the recombinant protein. Furthermore, experiments were conducted to verify that directly transfecting a CHO cell recombinant protein expression system with let-7a mimics can increase the expression level of a target protein, such as adalimumab. Furthermore, a let-7a overexpression vector was constructed by inserting a nucleic acid sequence encoding let-7a mimics into a starting vector, and the let-7a overexpression vector was transfected into a CHO cell recombinant protein expression system. Experiments demonstrated that increasing the amount of miRNA let-7a more efficiently increased the expression level of the target protein.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the use of let-7a in the expression of recombinant proteins in CHO cells and expression systems. [Background technology]

[0002] Recombinant antibody drugs have become a hot topic in drug research and development due to their strong targeting and minimal side effects. The use of Chinese hamster ovary (CHO) cells to express therapeutic recombinant protein drugs (antibodies) is currently an important platform for biopharmaceutical research, development, and production. However, low expression levels have traditionally been a key factor limiting the mass production of recombinant protein drugs. In cell culture, nutrient depletion, accumulation of toxic metabolites, and increased osmotic pressure can lead to cell apoptosis, severely impacting viable cell density and the yield and quality of the desired product. Through the construction of highly efficient expression vectors, host cell line modification, and production process optimization, recombinant protein expression levels can be effectively increased, improving cell productivity and maintaining high viable cell densities for extended periods.

[0003] MicroRNAs (miRNAs) are a type of evolutionarily conserved single-stranded non-coding RNA that are involved in post-transcriptional gene regulation by binding to target gene mRNAs and inducing target gene mRNA degradation or translational repression. Currently, the expression and biological functions of miRNAs in CHO cells have attracted widespread attention and can be used as potential cell therapy or engineering targets to enhance cellular productivity and stress tolerance. Compared with most methods that rely on gene overexpression or gene knockout, miRNAs can simultaneously regulate multiple different cellular pathways to maintain cellular homeostasis. By overexpressing or inhibiting specific miRNAs, the goal of simultaneously regulating multiple pathways, such as protein synthesis and secretion and cellular metabolism, can be achieved, resulting in increased production.

[0004] The let-7 family of miRNAs, one of the first discovered in Caenorhabditis elegans, is involved in the regulation of various physiological and pathological processes, including cell proliferation, differentiation, apoptosis, immune responses, tumorigenesis, and metastasis. There are two ways in which let-7 regulates target genes. When let-7 binds to the 3'UTR of target gene mRNA with perfect complementarity, it can directly cleave and degrade the target gene mRNA. However, when let-7 binds to the 3'UTR of target gene mRNA with imperfect complementarity, it can suppress post-transcriptional translation. Research has shown that the let-7 family, which is involved in the apoptosis pathway, can be used as a potential target for cell engineering. Studies on the expression of recombinant proteins in CHO cells using let-7a have not yet been reported.

[0005] Although the current production of recombinant proteins has reached a high level, with the continuous development of the biopharmaceutical industry, the demand for recombinant proteins produced by mammalian cells is increasing, and further increasing the production of mammalian cells has become an urgent issue that needs to be resolved. Summary of the Invention [Problem to be solved by the invention]

[0006] To overcome the drawbacks of the prior art, one of the objectives of the present invention is to provide a use of let-7a in CHO cell recombinant protein expression, which increases the amount of miRNA let-7a, thereby increasing the expression level of the recombinant protein.

[0007] A second object of the present invention is to provide a recombinant protein expression system that increases the amount of recombinant protein expressed by constructing CHO cells that highly express let-7a. [Means for solving the problem]

[0008] In order to achieve the above object of the invention, the technical solutions adopted in the present invention are as follows:

[0009] The present invention provides the use of let-7a in CHO cell recombinant protein expression, which increases the amount of miRNA let-7a having the nucleotide sequence set forth in SEQ ID NO. 1 in the CHO cell recombinant protein expression system.

[0010] Optionally, the amount of said miRNA in a CHO cell recombinant protein expression system is increased by transfecting the CHO cell recombinant protein expression system with let-7a mimics.

[0011] Specifically, the transfection amount of the let-7a mimics is 30 nM.

[0012] Optionally, the amount of said miRNA in a CHO cell recombinant protein expression system is increased by transfecting the CHO cell recombinant protein expression system with a let-7a overexpression vector.

[0013] Optionally, the let-7a overexpression vector is an expression vector comprising the let-7a mimics coding sequence shown in SEQ ID NO.2.

[0014] Let-7a mimics were constructed by transfecting CHO cells into a recombinant protein expression system. or constructed by transfecting a let-7a overexpression vector into a CHO cell recombinant protein expression system; let-7a mimics, let-7a overexpression vectors increase the amount of miRNA in a recombinant protein expression system, wherein the miRNA is cgr-let-7a having the nucleotide sequence shown in SEQ ID NO.1.

[0015] Specifically, the transfection amount of the let-7a mimics is 30 nM, Alternatively, the let-7a overexpression vector is an expression vector containing the let-7a mimics coding sequence shown in SEQ ID NO.2.

[0016] Optionally, the recombinant protein expression system is used to produce a formulation comprising the protein of interest; The preparation is selected from a protein detection reagent, a target protein drug used for treating or preventing a disease, and a gene drug having a target protein.

[0017] For example, in a specific embodiment of the present invention, the recombinant protein is adalimumab.

[0018] The let-7a described in the present invention is cgr-let-7a. [Effects of the Invention]

[0019] The beneficial effects of the present invention are as follows:

[0020] In this invention, increasing the amount of miRNA let-7a in a CHO cell recombinant protein expression system increases the expression level of the recombinant protein. Furthermore, experiments were conducted to verify that direct transfection of let-7a mimics into a CHO cell recombinant protein expression system can increase the expression level of a target protein, such as adalimumab. Furthermore, a let-7a overexpression vector was constructed by inserting a nucleic acid sequence encoding let-7a mimics into a starting vector, and the let-7a overexpression vector was transfected into a CHO cell recombinant protein expression system. Experiments demonstrated that increasing the amount of miRNA let-7a more efficiently increased the expression level of the target protein. The present invention provides a novel strategy for improving CHO cell recombinant protein expression. [Brief explanation of the drawings]

[0021] [Figure 1] 1 shows Western blot detection of the effect of transfection of let-7a mimics on the expression of adalimumab in Example 1. [Figure 2] 1 shows ELISA detection of the effect of transfection of let-7a mimics on the expression level of adalimumab in Example 1. [Figure 3] FIG. 1 shows a let-7a overexpression vector in Example 2. [Figure 4] 1 shows qPCR detection of let-7a expression levels in let-7a stably expressing CHO cell pools in Example 2. [Figure 5] 1 shows ELISA detection of the effect of stable overexpression of let-7a on the expression level of adalimumab in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0022] Unless otherwise specified, the relevant procedures in the examples and test examples are conventional techniques in the art. The various media, reagents, starting vectors, cell lines, tool enzymes, etc. used in the examples and test examples are all commercially available products.

[0023] The CHO cells used in the following examples are CHO-S cells purchased from Gibco.

[0024] The let-7a mimics and mimics NC used are purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0025] The adalimumab plasmid was independently constructed as follows: Using the Freedom® pCHO1.0 vector (Invitrogen, Thermo Fisher Scientific) as the starting vector, the adalimumab light chain (LC) and heavy chain (HC) sequences were cloned downstream of the EF2 / CMV and CMV / EF1 promoters, and a series of vector optimizations were performed to independently construct the pLC-pHC dual promoter eukaryotic expression vector.

[0026] Adalimumab-stably expressing CHO cells was independently constructed. The specific method was as follows: CHO cells were transfected with the above-mentioned adalimumab plasmid, and then screened for 2-3 weeks using blasticidin to obtain a cell pool. The monoclonal cell line was then screened using limiting dilution to obtain adalimumab-stably expressing cells.

[0027] The following examples further illustrate the present invention without, however, limiting it in any way.

[0028] Example 1: Detection of the effect of let-7a mimics transfection on adalimumab expression (1) Cell transfection Adalimumab-stably expressing CHO cells were cultured to logarithmic growth phase in DMEM / F12 complete medium, and the day before transfection, the cells were plated at 5 × 10 per well. 5 Cells were seeded into a 12-well cell culture plate, 1 mL of DMEM / F12 was added to each well, and the plate was cultured for 24 hours in a 37°C, 5% CO2 incubator. The next day, when the cells reached 70%-80% confluence, they were divided into two groups and transfected with let-7a mimics and mimics NC, respectively.

[0029] The transfection steps are as follows: 1. 50 μl of DMEM / F12 basal medium and 30 nM let-7a mimics were added to a 1.5 mL centrifuge tube and mixed evenly to prepare a diluted mimics solution. Mimics NC was used as a control. 2. Another 1.5 mL centrifuge tube was prepared, and 50 μl of DMEM / F12 basal medium and 2.0 μl of Lipofectamine 2000 were added and mixed uniformly to prepare a diluted transfection reagent solution, which was then allowed to stand at room temperature for 5 minutes. 3. The transfection reagent diluted in step 2 was added to the mimcs diluted solution in step 1, mixed gently to homogenize, and incubated at room temperature for 20 minutes to prepare the transfection complex. 4. The transfection complex was added dropwise to the cell culture plate, and the culture plate was gently shaken to mix evenly. 5. The culture plate was placed in a 37°C, 5% CO2 incubator and cultured for 4 to 6 hours, after which the medium was replaced and the culture was continued for 48 hours.

[0030] (2) Cell suspension culture 48 h after transfection, the cells were digested with trypsin to obtain a single cell suspension. 1 mL of serum-free CHO cell medium (purchased from Henan PuNuoYi Bioproducts Research Institute Co., Ltd.) was added to resuspend the cells. The cell suspension was mixed uniformly with 0.2% trypan blue solution at a 1:1 ratio and added to a cell counting plate. The viable cell density was detected using a Countstar Rigel S2 cell counter. The viable cell density was 5 × 10 5 The cells were seeded at 1000 cells / mL into a 12-well cell culture plate, and 2 mL of serum-free medium (purchased from Henan PuNuoYi Biological Products Research Institute Co., Ltd.) was added to each well. The culture plate was placed on a shaker in a 5% CO2 incubator at 37°C and 120 rpm for suspension culture.

[0031] (3) Detection of adalimumab expression levels After 7 days of suspension culture, the cell suspension was collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was collected and boiled for 10 minutes in 5x protein loading buffer. Adalimumab expression was detected by Western blot analysis. As shown in Figure 1, the expression level of adalimumab in the let-7a mimics transfection group was higher than that in the mimics NC transfection group. The cell culture supernatant was collected and adalimumab expression levels were detected in the supernatant using a human immunoglobulin G1 enzyme-linked immunosorbent assay kit (Kolu Biotechnology Co., Ltd.) according to the manufacturer's instructions. As shown in Figure 2, the adalimumab expression levels in the let-7a mimics transfection group and the mimics NC transfection group were 8.20 μg / mL and 13.51 μg / mL, respectively. Compared to the control group (mimics NC), the adalimumab expression level in the let-7a mimics transfection group was increased by 1.65-fold. This indicates that upregulating let-7a expression can increase adalimumab expression.

[0032] Example 2 Construction of let-7a overexpressing CHO cells (1) Construction of let-7a overexpression vector Using the pGCMV / EGFP / miR / Blasticidin vector (Shanghai Jima Pharmaceutical Technology Co., Ltd.), the let-7a mimics coding sequence was inserted downstream of the EGFP reporter gene of the starting vector to construct the let-7a overexpression vector pGCMV-let-7a mimics. pGCMV-NC served as the control vector. The let-7a mimics coding sequence is shown in SEQ ID NO. 2, and the vector map is shown in Figure 3.

[0033] (2) Screening of stable cell pools The day before transfection, 5 × 10 CHO cells in logarithmic growth phase were cultured per well. 5Cells were seeded into 12-well cell culture plates at a volume of 1000 x 1000 cells. 1 mL of DMEM / F12 complete medium was added to each well and cultured overnight in a 37°C, 5% CO2 incubator. When cell density reached 70%-80%, the cells were transfected with the overexpression vector pGCMV-let-7a mimics and the control vector pGCMV-NC using Lipofectamine 2000 transfection reagent. 48 h after transfection, 15 μg / mL blasticidin was added for screening. Once all untransfected cells had died, screening was continued for 2-3 weeks using 10 μg / mL blasticidin to obtain a pool of CHO cells with stable high let-7a expression (transfected with the overexpression vector pGCMV-let-7a mimics) and a control group of CHO cells (transfected with the control vector pGCMV-NC).

[0034] (3) qPCR detection of let-7a expression levels Design and synthesis of let-7a detection primers Forward primer: 5'-GGGCGTGAGGTAGTAGGTTGT-3'(SEQ ID NO.3) Reverse primer: 5'-AGTGCAGGGTCCGAGGTATT-3'(SEQ ID NO.4) RT Primer: 5' -GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT-3'(SEQ ID NO.5) RTPrimer:5' -GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT-3'(SEQIDNO.5) Total RNA was extracted from let-7a stably overexpressing CHO cells and control cells using Trizol. Total RNA was reverse transcribed into cDNA using a miRNA cDNA first-strand synthesis kit (Ikeguru Biotechnology Co., Ltd.). qPCR reactions were performed according to the instructions in the SYBR Green Pro Taq HS Premix qPCR kit. The qPCR reaction system consisted of 10 μl of 2×SYBR Green Pro Taq HS Premix (ROX plus), 2 μl of cDNA, 0.4 μl of Primer F, 0.4 μl of Primer R, and 7.2 μl of RNase-free water. The reaction conditions were 95°C for 30 s, 95°C for 5 s, 60°C for 30 s, 40 cycles, 95°C for 15 s, and 60°C for 1 min. -△△Ct Let-7a expression levels were calculated based on the

[0035] As shown in Figure 4, the expression level of let-7a in CHO cells with stable high let-7a expression (transfected with the overexpression vector pGCMV-let-7a mimics) was significantly higher than that in control cells (transfected with the control vector pGCMV-NC). Compared to the control group, the expression level of let-7a increased 8.22-fold.

[0036] Example 3: Detection of the effect of stable overexpression of let-7a on the expression level of adalimumab (1) Cell transfection The let-7a stable, highly expressing CHO cells constructed in Example 2 and the control cells were cultured at 5 × 10 per well. 5 Cells were seeded into 12-well cell culture plates at a dose of 1 × 10 cells and cultured overnight in a 37°C, 5% CO2 incubator. When the cell density reached 70%-80%, the adalimumab plasmid was transfected using Lipofectamine 2000 transfection reagent. 48 h after transfection, the cells were washed with PBS, digested with trypsin, and counted at 5 × 10 cells. 5The cells were seeded into a 12-well cell culture plate at a viable cell density of 1000 cells / mL, 2 mL of serum-free CHO cell medium was added to each well, and the culture plate was placed on a shaker in a 5% CO incubator at 37°C and 120 rpm for suspension culture.

[0037] (2) ELISA detection of adalimumab expression levels After 7 days of cell suspension culture, the cell suspension was collected in a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes, the cell supernatant was collected, and the expression level of adalimumab in the supernatant was detected by ELISA. As shown in Figure 5, the expression levels of adalimumab in the let-7a stable overexpressing CHO cells and the control cells were 21.69 μg / mL and 9.50 μg / mL, respectively. Compared with the control group, the expression level of adalimumab in the let-7a stable overexpressing CHO cells was increased by 2.28-fold. This indicates that stable overexpression of let-7a can significantly increase the expression level of adalimumab.

[0038] It should be noted that the above embodiments are only used to explain the technical solutions of the present invention, and do not limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art may modify the technical solutions recorded in the above embodiments or make equivalent substitutions for some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of each embodiment of the present invention.

Claims

1. Use of let-7a in CHO cell recombinant protein expression, characterized by increasing the amount of miRNA let-7a having the nucleotide sequence shown in SEQ ID NO. 1 in the CHO cell recombinant protein expression system.

2. The use according to claim 1, characterized in that the amount of said miRNA in a CHO cell recombinant protein expression system is increased by transfecting the CHO cell recombinant protein expression system with let-7a mimics.

3. The use according to claim 2, wherein the transfection amount of the let-7a mimics is 30 nM.

4. The use according to claim 1, characterized in that the amount of said miRNA in a CHO cell recombinant protein expression system is increased by transfecting the CHO cell recombinant protein expression system with a let-7a overexpression vector.

5. The use according to claim 4, wherein the let-7a overexpression vector is an expression vector comprising the let-7a mimics coding sequence shown in SEQ ID NO.

2.

6. Let-7a mimics were constructed by transfecting CHO cells into a recombinant protein expression system; or constructed by transfecting the let-7a overexpression vector into a CHO cell recombinant protein expression system; A recombinant protein expression system characterized in that let-7a mimics and let-7a overexpression vectors increase the amount of miRNA in the recombinant protein expression system, and the miRNA is cgr-let-7a having the nucleotide sequence shown in SEQ ID NO.

1.

7. The transfection amount of the let-7a mimics is 30 nM; Alternatively, the recombinant protein expression system according to claim 6, wherein the let-7a overexpression vector is an expression vector containing the let-7a mimics coding sequence shown in SEQ ID NO.

2.

8. Used in the production of formulations containing target proteins, The use of the expression system according to claims 6 to 7, characterized in that the preparation is selected from protein detection reagents, target protein drugs used for the treatment or prevention of diseases, and gene drugs having target proteins.

9. The use according to claim 8, characterized in that the target protein is adalimumab.

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