Overexpression vector of Fos gene, CHO cell line of overexpression Fos and construction method of CHO cell line

Abstract

The invention discloses an overexpression vector of an Fos gene, an overexpression Fos CHO cell line and a construction method of the overexpression Fos CHO cell line, and relates to the technical field of genetic engineering, the overexpression vector of the Fos gene comprises a PB513B-Fos-Myc-T2A-Puro plasmid vector constructed by Fos and a CHO-Fos cell line prepared by overexpression Fos; the overexpression vector comprises a codon-optimized Fos gene coding sequence as shown in SEQ ID NO.2, and the codon-optimized Fos gene coding sequence is inserted into a eukaryotic expression vector. The CHO cell line is obtained by transfecting a carrier to CHO cells and carrying out puromycin screening and monoclonal treatment on the CHO cells. According to the method, global transcription is activated through Fos overexpression, the position effect is overcome, the expression quantity of the recombinant protein such as K6-mAb is remarkably improved, and an efficient and stable system is provided for recombinant protein production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a Fos gene overexpression vector, a Fos overexpression CHO cell line and a construction method thereof. BACKGROUND

[0002] Monoclonal antibody (mAb) drugs have developed rapidly in the past decade and have become one of the most popular biological drugs in the world, with a market value of more than 300 billion US dollars by 2025. At present, nearly 89% of the approved recombinant therapeutic antibodies (RTAs) are produced by Chinese hamster ovary (CHO) cells. CHO cells have become the preferred host cells for producing recombinant mAbs due to their stable growth, easy large-scale culture, low susceptibility to human viruses, and similar post-translational modifications to human cells.

[0003] However, efficient and stable expression of a gene of interest (GOI) in CHO cells is still a key technology. Since GOIs are usually inserted into the host genome by random integration, they are susceptible to the "position effect", which leads to transgene silencing or low expression levels, severely restricting the production of recombinant mAbs.

[0004] One of the strategies to improve the productivity of CHO cells is to use transcription factors. Transcription factors can simultaneously regulate the expression of multiple key genes, thereby improving cell growth conditions and enhancing protein expression capacity. Therefore, screening effective transcription factors and modifying CHO cell lines with them have important scientific research value and industrial application prospects for improving the production of recombinant proteins.

[0005] Fos gene (also known as c-fos or AP-1) is a proto-oncogene that encodes a transcription factor belonging to the basic leucine zipper (bZIP) family. As a key component of the AP-1 transcription complex, Fos can form dimers with proteins such as Jun, recognize and bind to specific DNA sequences, and then regulate the expression of a series of downstream genes. Although Fos plays an important role in various cellular processes, there is no research report on whether its overexpression in CHO cells can improve the production capacity of recombinant proteins. SUMMARY

[0006] The present application provides a Fos gene overexpression vector, a Fos overexpression CHO cell line and a construction method thereof, which solves the problem of low expression level caused by the position effect in the prior art, realizes the construction of Fos overexpression plasmid and transient expression, the integration of Fos into the CHO cell locus, the stable overexpression of Fos in CHO cells, and the improvement of the expression amount of recombinant proteins when applied to the construction of recombinant protein expression system, thereby providing a direction for the construction of CHO cells and the production of recombinant proteins.

[0007] The application provides a Fos gene overexpression vector, which is constructed by inserting a codon-optimized Fos gene coding sequence into a multiple cloning site of a eukaryotic expression vector PB513B-Myc-T2A-Puro plasmid, and the codon-optimized Fos gene sequence is SEQ ID NO. 2.

[0008] Further, the vector further comprises a CMV promoter, a Myc tag, a T2A "self-cleavage" peptide segment and a puromycin resistance gene.

[0009] A CHO cell line overexpressing Fos is obtained by transfecting the Fos gene overexpression vector into a CHO-S cell line and screening a CHO cell stably overexpressing Fos.

[0010] Further, the CHO cell line is obtained by screening with puromycin.

[0011] The method for constructing the CHO cell line overexpressing Fos comprises the following steps: Transfecting the overexpression vector into CHO cells; Screening with puromycin; Screening a single clone cell by limiting dilution method.

[0012] Further, the transfection is a liposome transfection method.

[0013] Further, the screening concentration of puromycin is 15 μg / mL.

[0014] The one or more technical solutions provided in the embodiments of the application have at least the following technical effects or advantages: In the application, the synthesized Fos coding gene nucleic acid sequence is cloned into a starting vector to construct a Fos overexpression vector, and the Fos overexpression vector is integrated into the gene of a CHO cell to realize stable overexpression of Fos in the CHO cell.

[0015] The Fos overexpression vector is applied to construct a recombinant protein expression system, the Fos overexpression vector is transfected into a CHO cell to construct a CHO cell line overexpressing Fos, the CHO cell line can significantly improve the expression level of Fos K6-mAb, and the CHO cell line is used as a host cell for recombinant protein expression, compared with a wild-type CHO cell line, the expression amount of recombinant K6-MAB is improved, and the problem of low expression level in the current CHO cell expression system is effectively overcome. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1A schematic diagram of a PB513B-Myc-T2A-Puro plasmid structure for overexpressing Fos gene according to the present application; Figure 2 A comparison chart of the effect of transient overexpression of Fos gene in CHO cell line according to the present application; Figure 3 A comparison chart of the effect of CHO cell line overexpressing Fos gene on enhancing K6-MAB expression according to the present application. DETAILED DESCRIPTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting upon the application; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0018] Embodiment One: The present embodiment provides a Fos gene overexpression vector; the overexpression vector is constructed by inserting the codon-optimized Fos gene coding sequence into a eukaryotic expression vector.

[0019] The Fos gene is regulated by the CMV promoter, and the nucleotide sequence of the Fos gene is shown in SEQ ID NO. 1; the specific sequence is: The Fos CDS region nucleic acid fragment sequence was synthesized by General Biosystems (Anhui) Co., Ltd. after sequence optimization, as shown in SEQ ID NO. 2. The Fos CDS region was then inserted into the multiple cloning site of the eukaryotic expression vector PB513B-Myc-T2A-Puro plasmid by molecular cloning technology to obtain a Fos overexpression vector containing a CMV promoter, a Myc tag, a T2A "self-cleavage" peptide segment, and a puromycin resistance gene. The successfully constructed plasmid was named PB513B-Fos-Myc-T2A-Puro, and the schematic diagram of the structure of the plasmid is shown in Figure 1 .

[0020] The specific sequence of the sequence-optimized Fos nucleic acid fragment is SEQ ID NO. 2: CHO-K6-mAb cells (constructed in the laboratory) were cultured in DMEM / F-12 medium containing 10% inactivated fetal bovine serum (purchased from Henan Punuoibiological Product Research Institute Co., Ltd.) at 37°C, 5% CO2, to the logarithmic growth phase, and 2.0 x 10 5 cells were seeded in a 12-well cell culture plate, and plated for 24 h. When the cell confluence reached about 85%, the cells were used for transfection.

[0021] The Fos-overexpressing vector (2.5 μg / well) constructed by lipofection was transfected into the CHO-K6-mAb cells obtained above to obtain Fos-overexpressing CHO cells as the overexpression (Fos) group. At the same time, the blank CHO-K6-MAB cells were transfected with empty vectors as the control group (Control).

[0022] The lipofection method is as follows: (1) Take a 1.5 mL centrifuge tube labeled with the vector name as an example, add 50 μL of basic DMEM / F-12 medium into the tube, then take 1 μg of the constructed PB513B-Fos-Myc-T2A-Puro plasmid vector and add it into the tube, mix well, and stand at room temperature for 5 min; (2) Take another centrifuge tube, label it as transfection reagent tube, add 50 μL of basic DMEM / F-12 medium into the tube, then add 2.5 μL of transfection reagent Lipofectamine 3000 into the tube, mix well, and stand at room temperature for 5 min; (3) Add the mixture in step (2) to the plasmid tube in step (1), mix well, and stand at room temperature for 20 min.

[0023] (4) Take the cells plated for transfection the day before from the incubator, discard the old medium, wash each well twice with 1 x PBS buffer, then add 500 μL of new DMEM / F12 medium without double antibodies; (5) Add the mixture in step (3) to the well, gently shake the culture plate to distribute the mixture evenly, and then place it in the incubator for incubation. After 4 h, take out the culture plate, replace it with fresh complete medium for continued culture; after 48 h of cell transfection, trypsinize and centrifuge, count with 0.08% trypan blue, and inoculate at a density of not less than 3.0 x 10 5 cells / mL, and detect the K6-mAb expression level after 7 days of suspension expression. As shown in Figure 2 , the results show that Fos transient transfection can significantly increase the expression level of recombinant K6-mAb by 2.06 times.

[0024] Example 2: The present application provides a CHO-Fos cell line, which is a CHO cell line overexpressing Fos. The CHO-S cell line (purchased from Gibco) is cultured in DMEM / F12 medium containing 10% inactivated fetal bovine serum at 37°C, 5% CO2, to the logarithmic growth phase, and then inoculated in a 12-well cell culture plate at a cell density of 2.0 x 10 5 5 The cells are cultured for 24 h, and then used for cell transfection when the cell confluence reaches about 85%.

[0025] The liposome transfection method is as follows: the cell transfection conditions are the same as in Example 1; 48 h after cell transfection, the optimal Purinomycin concentration (15 ug / mL) is used for cell screening. After drug treatment, if the cells do not die, the screening concentration is reduced, and the culture is expanded. The cells are collected and counted, and the single clone is screened by limiting dilution, 1 cell per well is inoculated in a 96-well plate, and the cells are allowed to proliferate. When the cell confluence in the 96-well plate reaches about 90%, the culture is expanded, and the subsequent passage and screening steps can be performed.

[0026] Recombinant protein expression of CHO-Fos cell line: the eukaryotic expression vector of K6-mAb monoclonal antibody constructed in the laboratory is transfected into Fos group and Control group CHO cells. 48 h after transfection, the recombinant CHO cells are cultured in a six-well plate with 3 mL of protein-free, serum-free, chemically defined CD CHO medium (Henan Puno Yi Biological Product Research Institute Co., Ltd.) for 6 d to a cell number of 1 x 10 7 7 The cell density and viability are detected by Countstar BioTech cell counter (Shanghai Ruiyu Biological Technology Co., Ltd.) every day, and the supernatant is collected by centrifugation on the 7th day, quantified with the same cell number, and used for detection and analysis of the expression level of recombinant K6-mAb monoclonal antibody. Western blot is used to detect and analyze the expression of K6-mAb monoclonal antibody in Fos group and Control group CHO cells. As shown in Figure 3 The results show that the Fos-overexpressing CHO cells can significantly increase the expression level of recombinant K6-mAb monoclonal antibody, and the expression level is increased by 2.51 times.

[0027] The above experiment uses the eukaryotic expression vector of K6-mAb monoclonal antibody to transfect CHO-Fos cell line and PB513B-Fos-Myc-T2A-Puro plasmid vector to transfect CHO-K6-MAB cells, thereby improving the expression of K6-mAb. The following is an analysis of the results. First, it is fundamentally that Fos is a transcription factor, which can globally increase the transcriptional activity of CHO cells, thereby increasing the expression level of exogenous genes. After Fos expression, Fos protein combines with Jun protein and other factors to form AP-1 transcription factor complex, which can recognize and bind to many specific DNA sequences in the promoter region of genes. After binding, other transcriptional auxiliary activation factors and histone modification enzymes are recruited, the compact chromatin structure becomes loose, and the assembly of the transcription initiation complex is promoted, thereby strongly activating gene transcription, so that a large number of AP-1 complexes are formed in the cell, which are distributed throughout the genome and bind to all target points they can contact. Even if the target gene is integrated into the originally silent heterochromatin region, this region may be affected by the AP-1 complex and the auxiliary activation factors and modification enzymes recruited by it due to the presence of AP-1 target points nearby, resulting in the opening of the chromatin structure, the reactivation of the originally consistent CMV promoter, and the driving of the target gene for efficient transcription.

[0028] While the CHO-Fos cell line has been able to stably express Fos, it is a purified and uniform cell population, providing a stable and uniform transcriptional activation background. However, after transfection of PB513B-Fos-Myc-T2A-Puro, it cannot ensure that the expression amount of each cell is consistent, and it will be diluted and degraded as the cell line grows, resulting in an expression amount that is higher but not as stable and total as the CHO-Fos cell line; The stably expressed Fos cell line has adapted to the metabolic and physiological changes brought about by Fos overexpression, and the cell has reached a new and more efficient balance between growth and expression; while transient transfection of Fos is a drastic and sudden disturbance to the cell state, the cell needs time to adapt, but by the time the experiment is measured, the expression of Fos may have already begun to decline.

[0029] However, both the PB513B-Fos-Myc-T2A-Puro plasmid vector and the CHO-Fos cell line have high-efficiency transcription through the Fos gene, through the global transcriptional activator Fos, and through the transcription level of the entire cell genome environment to ensure that no matter where the GOI is integrated, there is a higher probability of being in an open and suitable state for transcription, thereby significantly overcoming the problem of transgene silencing and low expression level caused by "position effect", and ultimately achieving stable and high-yield recombinant mAb production.

[0030] In subsequent studies, it was found that the use of preheated culture medium and PBS solution before transfection can further promote expression, specifically, the eukaryotic expression vector transfection of K6-mAb monoclonal antibody in CHO-Fos cell line can further improve the expression by 7% (2.69 times increase); PB513B-Fos-Myc-T2A-Puro plasmid vector transfection of CHO-K6-MAB cell can further improve the expression by 21% (2.49 times increase); for plasmid vector transfection, preheating can increase the ability of transient expression, since the expression ability of Fos gene may gradually decrease over time after Fos plasmid transfection, therefore, providing the most suitable environment at the beginning can ensure the stability of cell state and accelerate the assembly of AP-1 complex, which has a greater improvement, but for the stable CHO-Fos cell line, the improvement is small (the expression ability of CHO-Fos cell line itself is already strong); In transient transfection, preheating helps cells quickly recover metabolism after transfection, promotes timely expression of Fos protein, thereby more effectively activates transcription of K6-mAb gene, also helps more cells to absorb plasmid, leading to short-term but efficient expression of Fos protein, and then activates global transcription through AP-1 transcription factor complex to improve K6-mAb expression; in stable cell line, preheating enables cells to adapt to metabolic changes caused by Fos overexpression more quickly after transfection, reduces cell stress, and helps cells to reach a new balance between growth and expression. For CHO-Fos cell line construction, preheating improves initial transfection efficiency, enables more cells to integrate Fos gene, and facilitates subsequent selection of stable clones by puromycin. This lays a foundation for obtaining uniform and high-expression CHO-Fos cell line.

[0031] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A Fos gene overexpression vector, characterized in that, The plasmid vector was constructed by inserting the codon-optimized Fos gene coding sequence into the multiple cloning site of the eukaryotic expression vector PB513B-Myc-T2A-Puro plasmid. The codon-optimized Fos gene sequence is SEQ ID NO.

2.

2. The Fos gene overexpression vector as described in claim 1, characterized in that, The vector also contains a CMV promoter, a Myc tag, a T2A "self-splicing" peptide, and a puromycin resistance gene.

3. A CHO cell line overexpressing Fos, characterized in that, The CHO-S cell line was obtained by transfecting the Fos gene overexpression vector as described in claim 1 into the CHO-S cell line and then screening for CHO cells that stably overexpress Fos.

4. The CHO cell line overexpressing Fos as described in claim 3, characterized in that, The CHO cell line was obtained through screening with puromycin.

5. A method for constructing a CHO cell line overexpressing Fos as described in claim 3, characterized in that, Includes the following steps: The overexpression vector was transfected into CHO cells; Screening was performed using puromycin; Monoclonal cells were screened using the limiting dilution method.

6. The method for constructing a CHO cell line overexpressing Fos as described in claim 5, characterized in that, The transfection method is liposome transfection.

7. The method for constructing a CHO cell line overexpressing Fos as described in claim 5, characterized in that, The screening concentration of puromycin was 15 μg / mL.

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