Application of miR-615-3p in regulating recombinant antibody expression in CHO cells

Abstract

The application provides an application of miR-615-3p in regulating recombinant antibody expression of CHO cells, and belongs to the technical field of biological medicine. The miRNA for regulating recombinant antibody expression of CHO cells is miR-615-3p, which can be targeted to combine with MEF2A, so as to regulate the expression. Overexpression of miR-615-3p can be used to increase the expression amount of recombinant antibody of CHO cells, the expression amount is increased by transfecting miR-615-3p mimics in a recombinant antibody expression system of CHO cells, or a CHO cell stably overexpressing miR-615-3p is constructed, so that the expression level of the recombinant antibody is improved. The application clearly shows the role of miR-615-3p in regulating recombinant antibody expression of CHO cells, and provides a potential target for engineering of CHO cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of miR-615-3p in regulating the expression of recombinant antibodies in CHO cells. Background Technology

[0002] Currently, approved recombinant antibody drugs occupy a core position in the recombinant protein drug market. Based on structural and functional differences, recombinant antibodies are mainly classified into monoclonal antibodies, antibody-drug conjugates, antibody fragments, bispecific antibodies, and antibody fusion proteins. Compared with small molecule chemical drugs, recombinant monoclonal antibodies, due to their highly specific targeting capabilities, can regulate disease-related signaling pathways and immune responses through multiple mechanisms such as ligand neutralization, apoptosis induction, and activation of immune effector functions, thereby playing a crucial role in disease treatment. Chinese hamster ovary (CHO) cells, possessing post-translational modification capabilities similar to human cells, have become a core platform for recombinant protein drug production. However, recombinant protein drug production still faces challenges such as low and unstable expression levels of the target protein, requiring further optimization strategies to improve the expression level of the target protein.

[0003] Modifying CHO cell lines using genetic engineering techniques can effectively improve cell viability and enhance recombinant protein expression and stability. Compared to most methods that rely on gene overexpression or knockout, miRNAs can regulate multiple cellular pathways, including transcription, translation, ribosome biosynthesis, secretion, cell cycle, metabolism, and cell death. Optimizing CHO cell lines by overexpressing or knocking down miRNAs to adapt to changes in stress, environment, and temperature can improve the yield and quality of recombinant antibodies. Previous studies have shown that miRNAs can influence recombinant antibody yield by regulating the expression of target genes; however, research on this is still limited and requires further investigation. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of miR-615-3p in regulating the expression of recombinant antibodies in CHO cells.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of miR-615-3p in regulating the expression of recombinant antibodies in CHO cells.

[0006] Preferably, the nucleotide sequence of miR-615-3p is shown in SEQ ID NO.1.

[0007] This invention provides the application of overexpression of miR-615-3p in enhancing the expression of recombinant antibodies in CHO cells.

[0008] Preferably, the method for enhancing the expression of recombinant antibodies in CHO cells includes: miR-615-3p mimics were transfected into a CHO cell recombinant antibody expression system; The nucleotide sequence of the positive strand of the miR-615-3p mimics is shown in SEQ ID NO.1; The nucleotide sequence of the antisense strand of the miR-615-3p mimics is shown in SEQ ID NO.2.

[0009] Preferably, the transfection amount of the miR-615-3p mimics is 20~50 nM.

[0010] Preferably, the method for enhancing the expression of recombinant antibodies in CHO cells includes: Transfect CHO cells with a recombinant expression vector containing the mature miR-615-3p sequence.

[0011] Preferably, the mature miR-615-3p sequence is shown in SEQ ID NO.3.

[0012] Preferably, the construction of the recombinant expression vector containing the mature miR-615-3p sequence includes ligating the mature miR-615-3p sequence into the LV19 vector.

[0013] Preferably, the recombinant antibody includes adalimumab or bevacizumab.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of miR-615-3p in regulating recombinant antibody expression in CHO cells. miR-615-3p can target and bind to MEF2A, thereby regulating its expression. Transfecting miR-615-3p mimics into a CHO cell recombinant antibody expression system enhances the expression level of recombinant antibodies. Simultaneously, ligating the mature miR-615-3p sequence into an LV19 vector to construct a miR-615-3p overexpression vector and transfecting it into CHO cells creates miR-615-3p overexpression cells, which serve as host cells for recombinant protein expression, further increasing the expression levels of recombinant antibodies such as adalimumab and bevacizumab. This invention clarifies the role of miR-615-3p in regulating recombinant antibody expression in CHO cells, providing a potential target for CHO cell engineering. Attached Figure Description

[0015] Figure 1 Western blot analysis was conducted to detect the effects of transfection with miR-615-3p mimics and inhibitors on adalimumab expression. Figure 2 To detect the effects of transfection with miR-615-3p mimics and inhibitors on miR-615-3p expression levels by qPCR; Figure 3 To detect the interaction between miR-615-3p and MEF2A in a dual-luciferase reporter gene assay; Figure 4 To observe cell fluorescence expression under a fluorescence microscope; Figure 5 To detect the expression level of miR-615-3p in the miR-615-3p stable cell pool using qPCR; Figure 6 To detect the effect of stable overexpression of miR-615-3p on adalimumab expression using Western blot; Figure 7 To detect the effect of stable overexpression of miR-615-3p on bevacizumab expression using Western blot. Detailed Implementation

[0016] This invention provides the application of miR-615-3p in regulating the expression of recombinant antibodies in CHO cells.

[0017] In this invention, the nucleotide sequence of miR-615-3p is shown in SEQ ID NO.1; SEQ ID NO. 1: 5'-UCCGAGCCUGGGUCUCCCUU-3'.

[0018] This invention provides the application of overexpression of miR-615-3p in enhancing the expression of recombinant antibodies in CHO cells.

[0019] In this invention, the method for enhancing the expression of recombinant antibodies in CHO cells includes: miR-615-3p mimics were transfected into a CHO cell recombinant antibody expression system. The miR-615-3p mimics are double-stranded RNA molecules formed by synthesizing a complementary strand based on the sequence of miR-615-3p; that is, double-stranded RNA molecules that chemically mimic miR-615-3p. The sequence of the sense strand is the same as SEQ ID NO. 1, and the sequence of the antisense strand is shown in SEQ ID NO. 2. SEQ ID NO.2: 5'-GAGGGAGACCCAGGCUCGGAUU-3'; The transfection amount of the miR-615-3p mimics is preferably 20~50 nM, more preferably 25~40 nM, and even more preferably 30 nM.

[0020] In this invention, the method for enhancing the expression of recombinant antibodies in CHO cells includes: CHO cells were transfected with a recombinant expression vector containing the mature miR-615-3p sequence. The mature miR-615-3p sequence is shown in SEQ ID NO.3. SEQ ID NO. 3: 5'-TCCGAGCTGGGTCTCCCTCTT-3'.

[0021] In this invention, the construction of the recombinant expression vector containing the mature miR-615-3p sequence includes ligating the mature miR-615-3p sequence into the LV19 vector.

[0022] In this invention, the recombinant antibody includes adalimumab or bevacizumab.

[0023] 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.

[0024] All operations described in this embodiment are conventional techniques in the field; the culture media, reagents, and materials used in this embodiment are all commercially available products.

[0025] The CHO cells used in this example were CHO-S, purchased from Gibco. miR-615-3p mimics and mimics NC, miR-615-3p inhibitor and inhibitor NC were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd. The nucleotide sequence of the miR-615-3p inhibitor is 5'-AAGAGGGAGACCCAGGCUCGGA-3' (SEQ ID NO.4). CHO cell serum-free culture medium was purchased from Henan Punoyi Biological Products Research Institute Co., Ltd. The HiScript III All-in-one RT SuperMix Perfect for qPCR kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. The ChamQ Universal SYBR qPCR Master Mix kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0026] Example 1

[0027] Detecting the effects of miR-615-3p mimics and inhibitors on the expression of recombinant antibodies in CHO cells

[0028] (1.1) Cell transfection

[0029] The revived adalimumab monoclonal cell line was cultured at 37°C until confluence reached approximately 90%, and the cells were digested with trypsin at a concentration of 5 × 10⁻⁶. 5 Cells were passaged in 12-well plates at a density of cells / mL.

[0030] The miR-615-3p mimics / NC and miR-615-3p inhibitor / NC dry powders were each prepared into 20μM solutions by adding 250μl of DEPC water.

[0031] When the cell confluence reached approximately 70%, the cells were replaced with fresh serum-free culture medium; then Lipofectamine was used. TM Cells were transfected using Transfection Reagent 2000 and divided into 5 groups: Control group (untreated group) and groups A, B, C, and D. Group A was the mimics NC group (transfected with mimics NC), Group B was the miR-615-3p mimics group (transfected with miR-615-3p mimics), Group C was the inhibitor NC group (transfected with inhibitor NC), and Group D was the miR-615-3p inhibitor group (transfected with miR-615-3p inhibitor).

[0032] Transfection steps: 1) Prepare two 1.5 mL centrifuge tubes. Add 50 μl of DMEM / F12 basal medium and 1.5 μl of miR-615-3p mimics solution to one tube and mix well to prepare a mimics dilution. Replace miR-615-3p mimics with mimicsNC in the other tube, leaving the rest unchanged, as a control. 2) Prepare two 1.5 mL centrifuge tubes. Add 50 μl of DMEM / F12 basal medium and 1.5 μl of miR-615-3p inhibitor solution to one tube and mix well to prepare the inhibitor dilution. Replace miR-615-3p inhibitor with inhibitor NC in the other tube, and keep everything else the same as the control. 3) Prepare four 1.5 mL centrifuge tubes and add 50 μl of DMEM / F12 basal medium and 1.5 μl of Lipofectamine to each tube. TM 2000, mix well to prepare a transfection reagent dilution solution, and let stand at room temperature for 5 minutes; 4) Add the diluted transfection reagent from step 3) to the mimics dilution solution from step 1), the inhibitor dilution solution from step 2), and the control, respectively, mix gently, and incubate at room temperature for 20 min; 5) Add the transfection complex to the cell culture plate and gently shake the plate to mix. 6) Place the culture plate in a 37℃, 5% CO2 incubator and incubate for 6 hours, then replace the culture medium and continue incubation.

[0033] (1.2) Western blot detection of recombinant antibody expression level

[0034] (1.2.1) Cell suspension

[0035] 48 hours after transfection, cells were digested with trypsin to form a single-cell suspension. Viable cell density was detected using a Countstar Rigel S2 cell counter at a concentration of 5 × 10⁶ cells / cells. 5 Live cells were seeded at a density of 1 / mL into 6-well cell culture plates, and 3 mL of serum-free CHO cell culture medium was added to each well. The culture plates were then placed in a 37°C, 5% CO2 incubator on a shaker at 120 rpm for suspension culture.

[0036] (1.2.2) Western blot

[0037] Cell suspension was collected, centrifuged at 1000 rpm for 5 min, and the supernatant was collected. 5× protein loading buffer was added, and the mixture was boiled for 10 min. Western blot was used to detect adalimumab expression levels. Results are as follows: Figure 1 As shown, miR-615-3p mimics can increase adalimumab expression levels, while miR-615-3p inhibitor groups show significantly reduced adalimumab expression levels. Compared to the control group, the miR-615-3p mimics transfection group showed a 1.43-fold increase in adalimumab expression levels, while miR-615-3p inhibitor transfection reduced adalimumab expression levels by 40%.

[0038] (1.2.3) Detection of miR-615-3p expression level by real-time quantitative PCR

[0039] Total RNA was extracted from cells in each group using FreeZol Reagent. RNA concentration and purity were measured using a NanoDrop 2000 ultra-micro spectrophotometer. Total RNA was reverse transcribed into cDNA using the HiScript III All-in-one RT SuperMix Perfectfor qPCR kit. qPCR reactions were performed according to the ChamQ Universal SYBR qPCR Master Mix kit instructions. Relative expression levels were calculated using a 2-1 ratio. -△△Ct Computational analysis was performed. The miR-615-3p specific primer sequence is as follows: RT Primer (SEQ ID NO.5): 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAGAGG-3'; Forward primer (SEQ ID NO.6): 5'-AACAAGTCCGAGCCTGGGTCT-3'; Reverse primer (SEQ ID NO.7): 5'-ATCCAGTGCAGGGTCCGAGG-3'; qPCR analysis results showed that, Figure 2 As shown, compared with the mimics NC group, the miR-615-3p expression level in the mimics transfected miR-615-3p group increased by 3.07 times; compared with the inhibitor NC group, the miR-615-3p expression level in the miR-615-3p inhibitor transfected group decreased by 52%.

[0040] (1.3) Dual-luciferase reporter gene assay

[0041] A fragment containing the predicted binding site in the 3' UTR of the artificially synthesized MEF2A gene was cloned into the NheI / XhoI restriction site of the dual-luciferase reporter vector pmirGLO to construct the wild-type reporter vector (MEF2A-WT). Simultaneously, a mutant reporter vector (MEF2A-MUT) was constructed using site-directed mutagenesis. Cells were co-transfected with the wild-type / mutant reporter vectors and miR-615-3p mimics / mimics NC, and divided into four groups: group A (MEF2A-WT + miR-615-3p mimics), group B (MEF2A-WT + mimics NC), group C (MEF2A-MUT + miR-615-3p mimics), and group D (MEF2A-MUT + mimics NC). Forty-eight hours after transfection, cells were collected, and luciferase activity was detected using the dual-luciferase reporter gene system.

[0042] The results are as follows Figure 3 As shown, miR-615-3p reduced luciferase activity in the wild-type MEF2A 3' UTR group, but had no significant effect on luciferase activity in the mutant 3' UTR group. These results indicate that miR-615-3p specifically inhibits luciferase activity in wild-type MEF2A 3' UTR, demonstrating that miR-615-3p can target and bind to MEF2A, thereby regulating its expression.

[0043] Example 2

[0044] Construction of miR-615-3p overexpressing CHO cells

[0045] (2.1) Construction of miR-615-3p overexpression vector

[0046] A DNA template sequence containing the mature miR-615-3p sequence was artificially synthesized and inserted into the CMV-GFP sequence of the LV19(CMV / GFP / IRES / Neo) vector to construct the overexpression vector LV19-miR-615-3p. LV19-NC served as the control vector.

[0047] (2.1.1) Carrier linearization

[0048] The LV19 vector was double-digested using restriction endonucleases Xho I and Not I. The digestion system consisted of 10 µl of 10×FastDigest Buffer, 5 µg of LV19 vector, 5 µl of Xho I, 5 µl of Not I, and ddH2O added to a final volume of 100 µl. Digestion was carried out at 37°C for 1 h. The results were detected by agarose gel electrophoresis. The correct bands were then selected for gel extraction. The concentration of the extracted products was determined using a nucleic acid analyzer and diluted to 50 ng / µl.

[0049] (2.1.2) Recombination reaction system

[0050] The vector ligation reaction was performed according to the following system: 2 µl 10×T4 Ligase Buffer, 1 µl LV19 linearized vector, 1 µl miR-615-3p DNA template, 1 µl T4 DNA Ligase, and 15 µl ddH2O. Ligation was carried out at 22℃ for 1 h. After the reaction, 5 µl of the solution was used to transform E. coli DH5α competent cells. Positive single clones were identified by colony PCR and sequenced for verification. Strains with correct sequencing results were extracted using a high-purity plasmid extraction kit.

[0051] (2.2) Construction of a stable cell pool

[0052] (2.2.1) Cell transfection

[0053] CHO cells were cultured to the logarithmic growth phase, and 5 × 10⁶ cells were cultured per well the day before transfection. 5 The cells were seeded into 12-well cell culture plates, with 1 mL of DMEM / F12 complete medium added to each well. The plates were then incubated at 37°C with 5% CO2 for 24 hours. When the cells reached 70%–80% confluence, Lipofectamine was used to... TMThe 2000 transfection reagent was used to transfect the overexpression vector LV19-miR-615-3p and the control vector LV19-NC, respectively.

[0054] (2.2.2) Screening stable cell pools

[0055] Forty-eight hours after transfection, 800 μg / mL G418 was added for selection. After all untransfected cells died, selection continued for 2-3 weeks using 600 μg / mL G418 to obtain miR-615-3p overexpressing CHO cells and control cells. Cell fluorescence was observed using an inverted microscope. Results are as follows: Figure 4 As shown, over 90% of the cells exhibit strong green fluorescence, indicating that a stable cell pool was successfully obtained through screening.

[0056] The expression level of miR-615-3p in the stable cell pool was detected by qPCR according to Example 1. The results are as follows: Figure 5 As shown, compared with the control group, the expression level of miR-615-3p in the stable cell pool increased by 15.28 times.

[0057] Example 3

[0058] Detecting the effect of miR-615-3p overexpression on recombinant antibody expression in CHO cells

[0059] (3.1) Cell transfection

[0060] The miR-615-3p overexpressing CHO cells constructed in Example 2 and control cells were seeded in 12-well cell culture plates. When the cell confluence was approximately 70%, Lipofectamine was used. TM 1.6 μg of adalimumab and 1.6 μg of bevacizumab plasmids were transfected using 2000 transfection reagent and divided into 5 groups, including the Control group (untransfected group) and groups A, B, C, and D. Group A was the LV19-NC group (control cells transfected with adalimumab plasmid), Group B was the LV19-miR-615-3p group (miR-615-3p overexpressing CHO cells transfected with adalimumab plasmid), Group C was the LV19-NC group (control cells transfected with bevacizumab plasmid), and Group D was the LV19-miR-615-3p group (miR-615-3p overexpressing CHO cells transfected with bevacizumab plasmid). 48 hours after transfection, 15 μg / mL of blast fungicide was added for selection. After all untransfected cells died, 10 μg / mL of blast fungicide was used for further selection for 2-3 weeks to obtain a stable cell pool of adalimumab and bevacizumab.

[0061] (3.2) miR-615-3p overexpression CHO cells

[0062] (3.2.1) Cell suspension

[0063] Culture each group of cells to the logarithmic growth phase, digest the cells with trypsin and count them, at a ratio of 5 × 10⁻⁶ cells / year. 5 Live cells were seeded at a density of 1 cell / mL into 6-well cell culture plates. 3 mL of serum-free CHO cell culture medium was added to each well. The culture plates were then placed in a 37°C, 5% CO2 incubator on a shaker at 120 rpm for suspension culture.

[0064] (3.2.2) Detection of recombinant antibody expression level

[0065] Cell culture supernatant was collected, and the expression levels of adalimumab and bevacizumab were detected by Western blot. Results are as follows: Figure 6 As shown, overexpression of miR-615-3p significantly increased the expression level of adalimumab, with a 96% increase compared to the control group. The results are as follows... Figure 7 As shown, compared with the control group, overexpression of miR-615-3p increased the expression level of bevacizumab by 118%. This indicates that CHO cells stably overexpressing miR-615-3p can serve as host cells to enhance the expression level of recombinant antibodies.

[0066] 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. Application of miR-615-3p in regulating the expression of recombinant antibodies in CHO cells.

2. The application according to claim 1, characterized in that, The nucleotide sequence of miR-615-3p is shown in SEQ ID NO.

1.

3. Application of miR-615-3p overexpression in enhancing recombinant antibody expression in CHO cells.

4. The application according to claim 3, characterized in that, The method for enhancing the expression of recombinant antibodies in CHO cells includes: miR-615-3p mimics were transfected into a CHO cell recombinant antibody expression system; The nucleotide sequence of the positive strand of the miR-615-3p mimics is shown in SEQ ID NO.1; The nucleotide sequence of the antisense strand of the miR-615-3p mimics is shown in SEQ ID NO.

2.

5. The application according to claim 4, characterized in that, The transfection amount of the miR-615-3p mimics is 20~50 nM.

6. The application according to claim 3, characterized in that, The method for enhancing the expression of recombinant antibodies in CHO cells includes: Transfect CHO cells with a recombinant expression vector containing the mature miR-615-3p sequence.

7. The application according to claim 6, characterized in that, The mature miR-615-3p sequence is shown in SEQ ID NO.

3.

8. The application according to claim 7, characterized in that, The construction of the recombinant expression vector containing the mature miR-615-3p sequence includes ligating the mature miR-615-3p sequence into the LV19 vector.

9. The application according to any one of claims 1 to 8, characterized in that, The recombinant antibody includes adalimumab or bevacizumab.

10. Application of miR-615-3p in inhibiting MEF2A activity.

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