Synthetic mar fragments, expression vectors, expression systems and uses thereof

Abstract

The present application relates to synthetic MAR fragments, expression vectors, expression systems and their applications, and belongs to the field of biotechnology. The novel synthetic MAR fragments are MAR-1, MAR-2 and MAR-3. The MAR fragments of the present application are designed and synthesized based on the common sequence characteristics of beta-globin MAR sequence, X-29 sequence, beta-interferon MAR sequence and MAR 1-68 sequence. It is found that when MAR-1, MAR-2 and MAR-3 are used simultaneously for the expression of exogenous proteins in CHO cells, the effect of MAR-1 is the best. The expression amount of exogenous proteins can be increased by 5.04-5.47 times compared with the system without MAR fragments. More preferably, the 8-copy tandem repeat sub-DNA fragment composed of MAR-1 fragment has better ability to increase the expression amount.

Description

Technical Field

[0001] This invention relates to artificially synthesized MAR fragments, expression vectors, expression systems, and their applications, belonging to the field of biotechnology. Background Technology

[0002] With the development of genetic engineering technology, the number and types of recombinant proteins produced using genetic engineering are constantly increasing, and have become an important part of the pharmaceutical industry. Recombinant drug protein expression systems mainly include E. coli, yeast, and non-humanized mammalian cell lines. E. coli is suitable for expressing proteins with small molecular weights and relatively simple structures, while yeast expression systems are suitable for expressing proteins with large molecular weights, more complex structures, and less glycosylation. Structural complexity or glycosylation is crucial for protein activity. Non-humanized mammalian cells possess post-translational modifications (PTMs) similar to human cells; therefore, mammalian cell expression systems are currently an important platform for the production of recombinant drug proteins. However, mammalian expression systems for recombinant protein production suffer from low yields and poor stability. The Chinese hamster ovary (CHO) cell expression system is currently the most widely used animal cell expression system, with nearly 70% of drug proteins (such as antibodies) produced using CHO cells.

[0003] However, CHO cell expression systems also suffer from drawbacks such as low target protein expression levels, long selection cycles for high-yield stable cells, and high cell culture costs, which severely restrict the production of recombinant protein drugs. Many factors influence the expression of recombinant proteins in CHO cells, with the expression vector being a crucial one. Furthermore, obtaining high-level, consistently stable cell lines for CHO cell expression is often difficult. This is because random integration after transfection silences or reduces the expression level of some transgenes. Therefore, it is necessary to isolate stable and highly efficient expression clones from a large number of cell clones, which poses a significant challenge to the industrial production of genetically engineered drugs (A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol. Bioeng. 2009).

[0004] The matrix attachment region (MAR) is the DNA sequence that remains attached to the nuclear matrix after restriction enzyme digestion. Studies have shown that MAR sequences can increase transgene expression levels in CHO expression systems while reducing the variability in transgene expression levels among transformants (Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat. Methods. 2007; Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells. Cell Biol. Int. 2010). However, MAR elements are relatively large. Commonly used MAR elements, such as the bicistronic expression vector, expression system, preparation method, and application disclosed in the invention patent with publication number CN106520832A, are all larger than 2000 bp in size. This increases the difficulty of vector construction, and the transfection efficiency of plasmids decreases with the increase of vector size, thus limiting the application of MAR elements in mammalian cell expression systems. Summary of the Invention

[0005] The purpose of this invention is to provide artificially synthesized MAR fragments.

[0006] This invention also provides applications of the aforementioned artificially synthesized MAR fragments.

[0007] The present invention also provides an expression vector comprising the above-described artificially synthesized MAR fragment.

[0008] This invention also provides applications of the above-mentioned expression vector.

[0009] The present invention also provides an expression system comprising the above-described expression vector.

[0010] The present invention also provides applications of the above-described expression system.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] The artificially synthesized MAR fragment has the nucleotide sequence shown in SEQ ID NO.1 (MAR-1), SEQ ID NO.2 (MAR-2), or SEQ ID NO.3 (MAR-3). Based on the analysis of the β-globin MAR sequence, X-29 sequence, β-interferon MAR sequence, and MAR 1-68 sequences, a novel MAR fragment was designed and synthesized according to their common sequence characteristics. Compared with the natural MAR sequence, it significantly improves the transgene expression level in CHO cells. The MAR-1 sequence is preferred. In this invention, it was found that when MAR-1, MAR-2, and MAR-3 are used simultaneously for the expression of exogenous proteins in CHO cells, MAR-1 shows the best effect. Compared with systems without the MAR fragment, the expression level of exogenous proteins can increase the cell pool of stable cell lines by 5.04-5.47 times.

[0013] The artificially synthesized MAR fragment of the present invention can be a tandem repeat DNA fragment composed of SEQ ID NO.1, SEQ ID NO.2, and / or SEQ ID NO.3. The preferred copy number is 2-16. More preferably, it is an 8-copy tandem repeat DNA fragment composed of the fragment shown in SEQ ID NO.1. Compared with a single copy number of MAR-1, EGFP is increased by 1.62 times, and compared with plasmids containing β-globin MAR sequences, it is increased by 2.44 times.

[0014] The aforementioned synthetic MAR fragments may also include DNA fragments containing transcription factor binding sites. The function of this fragment is to enhance transgene expression and transfection efficiency.

[0015] The novel artificially synthesized MAR sequence of this invention can be used to enhance the expression of transgenes in mammalian cell expression systems, while also improving the stability of recombinant protein expression, reducing expression differences between stable transformed cell lines, and achieving efficient, stable, and long-term expression of recombinant proteins in host cells.

[0016] An expression vector containing the aforementioned synthetic MAR fragment. The synthetic MAR fragment is inserted upstream of the promoter and / or downstream of the polyadenylation site in the expression vector. Preferably, the synthetic MAR fragment is inserted downstream of the polyadenylation site in the expression vector, at which position the expression level of the exogenous protein can be increased by 5.47 times compared to a system without the MAR fragment.

[0017] The above-mentioned expression vectors are used to express recombinant proteins. The specific application method is as follows: The artificially synthesized MAR sequence is inserted upstream of the promoter and / or downstream of the polyadenylation site of the mammalian cell expression vector; the exogenous gene clone is inserted into the expression vector before or after the MAR sequence insertion; the expression vector is transfected into mammalian host cells, and the mammalian cells are cultured to obtain the expressed target protein.

[0018] An expression system comprising the aforementioned expression vector. The host cells in the expression system are CHO, COS, HEK293, HT-1080, BHK, SP2 / 0, PER.C6, or C127 cells.

[0019] The above-mentioned expression system is used in the expression of recombinant proteins.

[0020] The artificially synthesized MAR fragment in this invention is inserted upstream of the promoter and / or downstream of the polyadenylation site of the expression vector. Compared with the vector without the artificially synthesized MAR fragment, the expression level of exogenous protein in the cell pool of stable CHO cells can be increased by more than 5 times, and by more than 2 times compared with the vector of the invention patent published in CN106520832A, thus achieving efficient, stable and long-term expression of recombinant protein in host cells. Attached Figure Description

[0021] Figure 1 This is a comparison of EGFP expression levels between expression vectors containing MAR-1, MAR-2, and MAR-3 and expression vectors containing β-globin MAR and those without MAR.

[0022] Figure 2 This is a comparison of EGFP expression levels between expression vectors containing MAR-1, MAR-4, MAR-5, MAR-6, and MAR-7 and expression vectors containing β-globin MAR and those without MAR.

[0023] Figure 3 This is a comparison of EPO expression levels between expression vectors containing MAR-1 and MAR-6 and expression vectors containing β-globin MAR and those without MAR.

[0024] Figure 4 This is a comparison of EPO expression levels in single-cell clones containing the MAR-6 fragment. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. The *Escherichia coli* JM109, pIRES-Neo plasmid vector, cell line reagents, and enzyme tools used in the embodiments and experimental examples are all commercially available. Endonucleases and NEBuffer were purchased from New England Biolabs LTD (NEB), USA, and the pIRES-Neo plasmid vector was purchased from Clontech Biotechnology.

[0026] Example 1

[0027] 1) Design and synthesis of artificial MAR fragments.

[0028] The MAR sequence is an AT-rich DNA sequence. Although it does not have a common sequence, the MAR sequence has typical characteristic motifs such as A-box, T-box, ARS, Unwinding sequence, Curved DNA, Oligo AT tracts, A-tracts, T-tracts, Stem loops, Curved DNA, SATB1, ATF site, Topoisomerase II, CEBP, FAST, Hox, NMP4, GSH, etc. (as shown in Table 1).

[0029] Table 1. Characteristic motifs of MAR

[0030]

[0031] In Table 1, Y represents T / C, W represents A / T, R represents G / A, N represents A / T / G / C, K represents G / T, S represents G / C, and M represents A / C.

[0032] According to literature reports (Genome-wide prediction of matrix attachment regions thatincrease gene expression in mammalian cells. Nat. Methods. 2007; Positional effects of the matrix attachment region on transgene expression in stablytransfected CHO cells. Cell Biol. Int. 2010);

[0033] (Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells. Gene. 2016). MAR sequences that enhance transgene expression are generally rich in motifs such as CEBP, FAST, GSH, Hox, and NMP4. Based on the above analysis, we designed and synthesized three MAR sequences: MAR-1 (i.e., sMAR-1, as shown in SEQ ID NO.1), MAR-2 (as shown in SEQ ID NO.2), and MAR-3 (as shown in SEQ ID NO.3). We also designed multiple copy sequences of MAR-1 as MAR-4 (2 copies, as shown in SEQ ID NO.4), MAR-5 (4 copies, as shown in SEQ ID NO.5), MAR-6 (8 copies, as shown in SEQ ID NO.6), and MAR-7 (12 copies, as shown in SEQ ID NO.7).

[0034] The MAR-1, MAR-2, MAR-3, MAR-4, MAR-5, MAR-6, and MAR-7 fragments were synthesized by General BioGene (Anhui) Co., Ltd.

[0035] Example 2

[0036] Construct an expression vector containing synthetic MAR-1 upstream of the CMV promoter.

[0037] To facilitate cloning, NruI (TCGCGA) and MluI (ACGCGT) restriction sites were inserted at the 5′ and 3′ ends of the synthesized MAR-1 fragment, respectively.

[0038] The synthesized MAR-1 sequence was digested with NruI / MluI, and the pIRES-Neo plasmid DNA vector (purchased from Clontech) was also digested with NruI / MluI. The digestion results were identified by agarose gel electrophoresis, and the digested MAR sequence fragment and pIRES-Neo linear plasmid DNA were recovered from the gel.

[0039] The double digestion system for the MAR-1 sequence was as follows: 10 μL (1 μg / μL) of MAR-1 sequence, 3 μL of 10×NE Buffer, 1.0 μL each of NruI and MluI (10 U / μL), and water was added to a final volume of 30 μL. The digestion conditions were: 37℃ for 3 min.

[0040] The double digestion system for pIRES-Neo plasmid was as follows: 5 μL pIRES-Neo plasmid (1 μg / μL), 3.12 μL 10×NEBuffer, 0.5 μL each of NruI and MluI (10 U / μL), and water was added to a final volume of 20 μL. The digestion conditions were: 37℃ for 3 min.

[0041] Take the enzyme-digested MAR-1 sequence fragment and pIRES-Neo linear plasmid DNA (molar ratio 5:1), and use NEB products. TM The ligation kit was used, and ligation was performed at 25°C for 5 min. The ligation product was added to a suspension of competent E. coli JM109 cells for transformation. 150 μL of the transformed bacterial culture was inoculated onto an LB agar plate containing ampicillin and incubated overnight at 37°C. Single colonies were picked for subculture. The recombinant plasmid was extracted and verified by double enzyme digestion (NruI / MluI). The plasmid that was correctly digested was sequenced for verification. The correctly constructed plasmid was named pIRES-MAR1-1.

[0042] Example 3

[0043] The construction method is the same as in Example 2, except that the fragment is replaced with the MAR-2 fragment (sequence shown in SEQ ID NO.2), and the correctly constructed plasmid is named pIRES-MAR1-2.

[0044] Example 4

[0045] The construction method is the same as in Example 2, except that the fragment is replaced with the MAR-3 fragment (sequence shown in SEQ ID NO.3), and the correctly constructed plasmid is named pIRES-MAR1-3.

[0046] Example 5

[0047] The construction method is the same as in Example 2, except that the fragment is replaced with the MAR-4 fragment (sequence shown in SEQ ID NO.4), and the correctly constructed plasmid is named pIRES-MAR1-4.

[0048] Example 6

[0049] The construction method is the same as in Example 2, except that the fragment is replaced with the MAR-5 fragment (sequence shown in SEQ ID NO.5), and the correctly constructed plasmid is named pIRES-MAR1-5.

[0050] Example 7

[0051] The construction method is the same as in Example 2, except that the fragment is replaced with the MAR-6 fragment (sequence shown in SEQ ID NO.6), and the correctly constructed plasmid is named pIRES-MAR1-6.

[0052] Example 8

[0053] The construction method is the same as in Example 2, except that the fragment is replaced with the MAR-7 fragment (the sequence is shown in SEQ ID NO.7), and the correctly constructed plasmid is named pIRES-MAR1-7.

[0054] Example 9

[0055] Construct an expression vector containing synthetic MAR-1 downstream of the promoter poly A.

[0056] To facilitate cloning, XhoI (CTCGAG) and BstZ17I (GTATAC) restriction sites were inserted at the 5′ and 3′ ends of the synthesized MAR-1 fragment, respectively. The synthesized MAR-1 sequence was then double-digested with XhoI / BstZ17I, and simultaneously...

[0057] pIRES-Neo plasmid DNA was digested with XhoI / BstZ17I. The digestion results were identified by agarose gel electrophoresis, and the digested MAR sequence fragment and pIRES-Neo linear plasmid DNA were recovered from the gel.

[0058] The double digestion system for the synthesized MAR sequence was: 10 μL (1 μg / μL) of MAR sequence. Add 3 μL of buffer, 1.0 μL each of XhoI and BstZ17I (10 U / μL), and bring the water volume to 30 μL. The enzyme digestion conditions are: 37℃, 3 min.

[0059] The double enzyme digestion system for pIRES-Neo plasmid is: 5 μL of pIRES-Neo plasmid (1 μg / μL). Add 2 μL of buffer, 0.5 μL each of XhoI and BstZ17I (10 U / μL), and bring the water volume to 20 μL. The enzyme digestion conditions are: 37℃, 3 min.

[0060] Agarose gel electrophoresis was used to identify the enzyme digestion results, and the digested MAR sequence fragments and pIRES-Neo linear plasmid DNA were recovered from the gel. (Using NEB equipment) TM The ligation kit was used, and ligation was performed at 25°C for 5 min. The ligation product was added to a suspension of competent E. coli JM109 cells for transformation. 150 μL of the transformed bacterial solution was inoculated onto an LB agar plate containing ampicillin and incubated overnight at 37°C. Single colonies were picked for subculture. The recombinant plasmid was extracted and verified by double enzyme digestion (XhoI / BstZ17I). The plasmid that was correctly digested was sequenced for verification. The correctly constructed plasmid was named pIRES-MAR2-1.

[0061] Example 10

[0062] The construction method is the same as in Example 9, except that the fragment is replaced with the MAR-2 fragment (sequence shown in SEQ ID NO.2), and the correctly constructed plasmid is named pIRES-MAR2-2.

[0063] Example 11

[0064] The construction method is the same as in Example 9, except that the fragment is replaced with the MAR-3 fragment (sequence shown in SEQ ID NO.3), and the correctly constructed plasmid is named pIRES-MAR2-3.

[0065] Example 12

[0066] The construction method is the same as in Example 9, except that the fragment is replaced with the MAR-4 fragment (sequence shown in SEQ ID NO.4), and the correctly constructed plasmid is named pIRES-MAR2-4.

[0067] Example 13

[0068] The construction method is the same as in Example 9, except that the fragment is replaced with the MAR-5 fragment (sequence shown in SEQ ID NO.5), and the correctly constructed plasmid is named pIRES-MAR2-5.

[0069] Example 14

[0070] The construction method is the same as in Example 9, except that the fragment is replaced with the MAR-6 fragment (sequence shown in SEQ ID NO.6), and the correctly constructed plasmid is named pIRES-MAR2-6.

[0071] Example 15

[0072] The construction method is the same as in Example 9, except that the fragment is replaced with the MAR-7 fragment (sequence shown in SEQ ID NO.7), and the correctly constructed plasmid is named pIRES-MAR2-7.

[0073] Comparative Example 1

[0074] In this comparative example, the expression vector does not contain MAR sequences upstream of CMV or downstream of the poly A promoter.

[0075] Comparative Example 2

[0076] Expression vectors containing β-globin MAR sequences upstream of CMV and downstream of the promoter poly A were constructed.

[0077] The method is described in the invention patent publication number CN106520832A. The carriers are named pIRES-MAR1-G and pIRES-MAR2-G.

[0078] Test case

[0079] The effect of synthetic MAR sequence on EGFP expression.

[0080] 1. Construction of expression vectors containing EGFP

[0081] 1) EGFP gene amplification

[0082] Primers P1 and P2 (for amplifying 720 bp EGFP gene DNA) were designed based on the Enhanced green fluorescent protein (EGFP) gene sequence of the pEGFP-C1 vector (GenBank: U55763.1, bases 613–1332). EcoRI and BamHI restriction enzyme sites were introduced at the 5′ ends of the primers, respectively. The primer sequences are shown below (underlined sites are restriction enzyme sites):

[0083] P1: 5′-CCG GAATTC ATGGTGAGCAAGGGCGAGGAG-3′;

[0084] P2: 5′-CTA GGATCCb GACTTGTACAGCTCGTCCATGC-3′.

[0085] The EGFP gene was amplified using pEGFP-C1 plasmid (purchased from Clontech, USA) as a template and primers P1 and P2. The PCR reaction system is shown in Table 2.

[0086] Table 2 PCR amplification system

[0087]

[0088] Reaction program: 95℃ for 3 min, 94℃ for 40 s, 56~60℃ for 30 s, 72℃ for 40 s, 4 cycles at each annealing temperature, and finally 55℃ for 1 min, 30 cycles, and 72℃ for 3 min.

[0089] The PCR amplification products were recovered by agarose gel electrophoresis, purified, and sent to a biotechnology company for sequencing verification. The results showed that the amplified DNA fragment was completely identical to the EGFP sequence published in GenBank.

[0090] 2) Construct expression vectors containing EGFP sequences

[0091] The PCR amplification product of EGFP (with sequences verified to be correct) was digested with EcoRI and BamHI. Simultaneously, the plasmids containing MAR-1, 2, 3, 4, 5, 6, and 7 from Examples 2-8, as well as the plasmids containing β-globin MAR sequences from Comparative Examples 1 and 2, were digested with EcoRI and BamHI. The digestion results were identified by agarose gel electrophoresis, and the digested EGFP sequence fragments and MAR-containing linear plasmid DNA were recovered from the gel.

[0092] The enzyme digestion system for the EGFP sequence was as follows: 2 μL of 10×M buffer, 0.5 μL each of 10 U / μL EcoRI and BamHI enzymes, 0.78 μL of 1.289 μg / μL EGFP amplification product, and water to a final volume of 20 μL. After thorough mixing, incubate at 37°C for 6 h.

[0093] The plasmid digestion system was as follows: 2 μL of 10×M buffer, 0.5 μL each of 10 U / μL EcoRI and BamHI enzymes, 1.23 μL of 0.81 μg / μL plasmid DNA, and water to a final volume of 20 μL. After thorough mixing, incubate at 37°C for 3 hours.

[0094] The digested EGFP sequence fragment and linear plasmid DNA were ligated using T4 ligase (ligation system: 10 μL 2×Quick Ligation Buffer, 200 ng pIRES-Neo2 linear plasmid DNA, 87.2 ng digested EGFP sequence fragment, 1 μL 350 U / μL T4 ligase, and water to a final volume of 20 μL), and incubated overnight at 16°C. The ligation product was then transformed into a suspension of competent E. coli JM109 cells. 100 μL of the transformed bacterial culture was inoculated onto an LB agar plate containing ampicillin and incubated overnight at 37°C. Single colonies were picked and cultured by shaking. Bacterial plasmids were extracted and recombinant plasmids were verified by enzyme digestion. Plasmids that were correctly identified by enzyme digestion were selected for sequencing verification.

[0095] 3) Cell transfection and screening of stably transfected cell lines

[0096] CHO cells were cultured at 37°C and 5% CO2 in DMEM medium containing 10% inactivated fetal bovine serum. CHO cells (3 × 10⁶ cells / well) were seeded into 6-well plates. 6 / well). After 24 hours of plate culture, the cells reached approximately 90% confluence. Using Lip3000 ( (3000) was used as the transfection reagent to transfect the expression vectors of each group into CHO cells. There were four experimental groups: ① Normal CHO cells; ② Control group – transfected with the control vector pIRES-EGFP without MAR; ③ Transfected with a vector containing the synthetic MAR sequence;

[0097] 48 hours later, 600 μg / mL of G418 was added to the transfection wells, and cell death occurred in large numbers starting from the fifth day. Two weeks after screening, the G418 concentration was adjusted to a maintenance concentration of 300 μg / mL for continued culture. After 30 days of culture, the polyclonal CHO cells obtained from the screening were collected from each experimental group for flow cytometry analysis.

[0098] See results Figure 1 , Figure 2 .Depend on Figure 1 It was found that, compared with the control vector pIRES-EGFP without MAR, expression vectors containing MAR-1, MAR-2, and MAR-3 all stably improved the stable expression level of the EGFP gene. The effect was more significant when the MAR fragment was upstream of the promoter than downstream of polyA. When MAR-1, MAR-2, and MAR-3 were located upstream of the promoter, the EGFP expression level increased by 5.04, 2.75, and 3.73 times, respectively, compared with Comparative Example 1; when located downstream of polyA, the increases were 5.47, 3.90, and 4.38 times, respectively. MAR-1 showed the most significant effect, with a maximum increase of 5.47 times (P<0.05). Compared with plasmids containing the β-globin MAR sequence, MAR-1 showed a 1.62-fold increase when located upstream of the promoter and a 1.59-fold increase when located downstream of polyA (P<0.05).

[0099] Experimental Example 2

[0100] To further screen for strong MAR elements, we compared the tandem repeats formed from multiple copies of synthesized MAR-1. MAR-1, 4, 5, 6, and 7 were inserted downstream of polyA. The results showed that as the copy number of MAR-1 elements increased, the EGFP expression level also increased. When the tandem copy number reached 8 copies (MAR-6), the EGPF gene expression level increased 8.58 times compared to the vector without MAR. Figure 2 (P<0.05). Compared with single-copy MAR-1, EGFP increased by 1.62-fold, and compared with plasmids containing the β-globin MAR sequence, it increased by 2.44-fold. Figure 2 (P<0.05).

[0101] Experimental Example 3

[0102] The effect of synthetic MAR sequences on EPO expression.

[0103] The specific steps for constructing a eukaryotic cell expression system are as follows:

[0104] 1. Construct expression vectors containing exogenous EPO genes

[0105] 1) Synthesize the EPO sequence

[0106] Based on the EPO sequence published by NCBI (GenBank: JN849371.1, bases 1-582), the artificial synthesis of the EPO sequence (with EcoRI and BamHI restriction sites introduced at the 5′ and 3′ ends, respectively), as well as the kozak sequence and signal peptide sequence, was carried out by General BioGene (Anhui) Co., Ltd.

[0107] 2) Construct expression vectors containing EPO sequences

[0108] The artificially synthesized EOP sequence was digested using EcoRI / BamHI double enzyme digestion primers. Simultaneously, pIRES-Neo2 and the MAR-1 and MAR-6 plasmid DNA from Examples 2 and 7 above were also digested with EcoRI / BamHI double enzymes. The digestion results were identified by agarose gel electrophoresis, and the digested EPO sequence fragments and linear plasmid DNA were recovered from the gel.

[0109] The double digestion system for the EPO sequence was as follows: 10 μL (1 μg / μL) of EPO sequence fragment, 3 μL of 10×NEBuffer 2.1, 1.0 μL each of EcoRI and BamHI enzymes (10 U / μL), and water was added to a final volume of 30 μL. The digestion conditions were: 37℃ for 3 min.

[0110] The double enzyme digestion system for the plasmid was as follows: 5 μL plasmid (1 μg / μL), 2 μL 10×NEBuffer 2.1, 0.5 μL each of EcoRI and BamHI (10 U / μL), and water was added to a final volume of 20 μL. The digestion conditions were: 37℃ for 3 min.

[0111] Take the enzyme-digested EPO sequence fragment and linear plasmid DNA (molar ratio 5:1), and use NEB (Nephrodisiac) for processing. TM The ligation kit was used, and ligation was performed at 25°C for 5 min. The ligation product was added to a suspension of competent E. coli JM109 cells for transformation. 150 μL of the transformed bacterial solution was inoculated onto an LB agar plate containing ampicillin and incubated overnight at 37°C. Single colonies were picked and subcultured. The recombinant plasmid was extracted and verified by double enzyme digestion (EcoRI / BamHI). The plasmid that was verified by enzyme digestion was used for sequencing verification.

[0112] 2. Construction of a eukaryotic cell expression system

[0113] Select healthy CHO cells and seed them onto 6-well culture plates. Transfection is performed when the plate density reaches approximately 80%. The specific steps are as follows: 10 μL of lipofectamine 2000 + 240 μL of serum-free Opti-MEM medium is added and incubated at 37°C for 5 min. Then, 250 μL (5 μg) of expression vector EPO is mixed with the serum-free Opti-MEM medium and incubated at 37°C for 20 min. Simultaneously, the cells on the 6-well culture plate are washed three times with PBS, and 2 mL of serum-free DMEM cell culture medium is added. Next, the mixture of liposomes and EPO plasmid DNA is gently added dropwise to the wells, and the plate is quickly and gently shaken to mix. The plate is then placed in a 5% CO2 cell culture incubator and cultured at 37°C for 6 h. After this, the serum-free DMEM medium is replaced with complete DMEM medium, and the plate is placed in a cell culture incubator for further culture. 48 hours later, 600 μg / mL of G418 was added to the transfection wells, and the medium was replaced with fresh D / F intact medium every 48 hours. From the fifth day onwards, a large number of cells began to die. After two weeks of selection, the G418 concentration was adjusted to a maintenance concentration of 300 μg / mL and cultured for another two weeks. After drug selection, a stable cell pool formed approximately two weeks later. Cells were cultured for another six days, and the cell supernatant was then used for ELISA detection.

[0114] The results showed that the EPO expression level using the expression system of this invention was significantly higher than that of traditional expression systems. The average EPO expression levels in CHO-S cells transfected with expression vectors MAR-1 and sMAR-6 plasmid DNA were 105.12 mg / L and 271.05 mg / L, respectively, while the EPO expression level in the pIRES-Neo2 expression system was only 31.24 mg / L, and the EPO expression level containing the β-globin MAR sequence was 106.53 mg / L (see...). Figure 3 ).

[0115] Simultaneously, after drug screening, the cells were subjected to limiting dilution monoclonalization. After approximately two weeks, a stable monoclonal cell pool was formed. The cells were then cultured for another 6 days, and the cell supernatant was collected for ELISA detection. The results are shown below. Figure 4 .Depend on Figure 4 It can be seen that among the 10 clones selected from CHO-S cells transfected with MAR-6 plasmid DNA, clones #2 and #8 had the best yields, at 404.23 mg / L and 374.51 mg / L, respectively.

Claims

1. An artificially synthesized MAR fragment, characterized in that: the nucleotide sequence shown in SEQ ID NO. 1, or a tandem repeat DNA fragment consisting of 2-8 copies of the nucleotide sequence shown in SEQ ID NO.

1.

2. Artificial synthetic MAR fragment according to claim 1, characterized in that: a tandem repeat DNA fragment consisting of 8 copies of the nucleotide sequence shown in SEQ ID NO.

1.

3. Artificial synthetic MAR fragment according to claim 1 or 2, characterized in that: Also included is a transcription factor binding site DNA fragment.

4. Use of the artificial synthetic MAR fragment according to any one of claims 1-3 for increasing the expression level of a recombinant protein in a mammalian cell expression system.

5. An expression vector comprising the artificial synthetic MAR fragment according to any one of claims 1-3.

6. The expression vector of claim 5, wherein: The artificial synthetic MAR fragment is inserted upstream of a promoter or downstream of a polyadenylation site in the expression vector.

7. Use of the expression vector according to claim 5 for increasing the expression level of a recombinant protein in a mammalian cell expression system.

8. An expression system comprising the expression vector of claim 5, characterized in that: The host cell in the expression system is a CHO, COS, HEK293, HT-1080, BHK, SP2 / 0, PER.C6 or C127 cell.

9. Use of the expression system according to claim 8 for expressing a recombinant protein.

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