A method for improving the expression level of recombinant protein in CHO cells

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a method for improving the expression level of recombinant proteins in CHO cells. The method comprises the following steps: (1) combining a target gene recombinant vector with a gene containing cell cycle regulation-related genes; p18 and / or genes that regulate cell growth aFGF The co-transgenic recombinant vector is co-transfected into CHO cells; the gene p18 The sequence of is shown in SEQ ID NO: 1; the gene aFGF The sequence is shown in SEQ ID NO:2; (2) co-transfecting the co-transgenic recombinant vector and the target gene recombinant vector into CHO cells, culturing and screening to obtain a CHO cell expression system; (3) culturing the transient and stable transfected CHO cell lines in suspension serum-free culture to obtain the target protein. The method of the present invention can significantly improve the expression level of the target gene in CHO cells and achieve transient and stable high-level expression of the target protein in CHO cells.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to a method for improving the expression level of a recombinant protein in a CHO cell. Background Art

[0002] With the development of genetic engineering technology, the demand for recombinant proteins in basic research, biotechnology applications and clinical treatment continues to grow. Mammalian cells are the preferred expression cells for recombinant drug proteins, among which Chinese hamster ovary cells (CHO) have the advantages of large-scale suspension growth suitable for industrial production, low risk of viral infection, product post-translational modification function, and the ability to produce high-quality proteins similar to human natural proteins and efficient amplification and expression of recombinant genes. They have become the most widely used mammalian cell line in industrial production.

[0003] In recent years, researchers have done a lot of work around improving the expression of recombinant proteins in CHO cells. For example, the productivity of CHO cells has been improved to a certain extent through methods such as optimization of culture medium, optimization of expression vectors, and transformation of cell lines. For example, the Chinese invention patent application with application publication number CN107828738A discloses a DNA methyltransferase-deficient CHO cell line and its preparation method and application, which achieves the problem of improving the expression level of recombinant protein and overcoming the problem of unstable expression of recombinant protein by knocking out the CHO cell DNA methyltransferase Dnmt3a gene. The Chinese invention patent application with application publication number CN109486859A discloses artificially synthesized polyA, mammalian cell recombinant expression vector, mammalian host cell, expression method and application, which increases the yield of recombinant protein in CHO cells by replacing the polyA sequence of the expression vector. The Chinese invention patent with application publication number CN110894487A discloses a serum-free and protein-free CHO cell culture medium and its preparation method and application, which promotes the growth of CHO cells and increases the yield of target protein by optimizing the culture medium components.

[0004] In addition to the optimization and transformation of culture media, expression vectors and cell lines, the production of recombinant proteins is also affected by protein translation, protein folding and secretion processes. The expression of recombinant proteins can be improved by overexpressing, downregulating or knocking out specific genes to improve or regulate processes such as apoptosis, cell cycle, protein folding and secretion. Summary of the invention

[0005] The purpose of the present invention is to provide a method for increasing the expression level of recombinant proteins in CHO cells. The method co-transfects a co-transgenic recombinant vector containing a gene regulating cell cycle and / or promoting cell growth and a target gene recombinant vector to regulate the expression of the target gene, thereby increasing the expression level of the recombinant protein in the CHO cells.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A method for improving the expression level of a recombinant protein in a CHO cell comprises the following steps:

[0008] (1) Recombining the target gene recombinant vector and the cell cycle regulation-related gene p18 Sequence and regulation of cell growth genes aFGF The co-transgenic recombinant vectors of the sequences were co-transfected into CHO cells;

[0009] Among them, p18 is a cyclin-dependent protein kinase inhibitor that can inhibit cyclin-dependent kinases, block cells in the G1 / S phase, and affect the expression of recombinant proteins by regulating the cell cycle. p18 The sequence of the gene is shown in SEQ ID NO:1.

[0010] Acidic fibroblast growth factor (aFGF), which promotes cell proliferation, affects the expression level of recombinant proteins by promoting cell proliferation. aFGF The sequence of the gene is shown in SEQ ID NO:2.

[0011] The co-transgenic recombinant vector and the target gene recombinant vector need to be transfected in a certain ratio. The present invention has found through screening that the optimal co-transfection ratio of the target gene recombinant vector and the co-transgenic recombinant vector is 10:1.

[0012] (2) The co-transgenic recombinant vector and the target gene recombinant vector are co-transfected into CHO cells, cultured and screened to obtain a CHO cell expression system; the CHO cell expression system of the present invention utilizes the co-transgenic recombinant vector containing the cyclin-dependent kinase inhibitor p18 and / or the acidic fibroblast growth factor aFGF to promote the efficient expression of the target gene, thereby increasing the expression amount of the recombinant protein in the CHO cells.

[0013] (3) Transiently and stably transfected CHO cell lines were cultured in suspension without serum to obtain the target protein.

[0014] In some specific embodiments, the CHO cell is any one of CHO-K1, CHO-S, and DG44. The CHO cell expression system prepared using CHO-K1, CHO-S, and DG44 cell lines can achieve corresponding technical effects and realize high-level transient and stable expression of the target protein.

[0015] In some specific embodiments, the transfection method is calcium phosphate method, electroporation, liposome transfection or PEI transfection.

[0016] In some specific embodiments, the target gene recombinant vector in step (1) is a eukaryotic expression vector containing an EGFP reporter gene, an adalimumab gene or a vitronectin (VTN) gene.

[0017] In some specific embodiments, the co-transgenic recombinant vector in step (1) is constructed based on the pIRES-neo vector. p18 , aFGF , p18 + aFGF or aFGF + p18 Eukaryotic expression vectors such as Figure 1 , 2 , 3. Regarding the resistance gene, in the present invention, the resistance gene of the co-transformation expression vector is replaced with blasticidin resistance, so that the selection markers of the target gene recombinant vector and the co-transformation recombinant vector are different.

[0018] When the co-transgenic recombinant vector contains p18 and aFGF When two genes p18 Genes can be located in aFGF Upstream or downstream of the gene, as a preferred solution, p18 Gene located in aFGF When the target protein is upstream of the gene, the expression level of the target protein is higher.

[0019] The above preparation method can be used to prepare a CHO cell expression system, and then the stably transfected CHO cell line is subjected to serum-free suspension culture to achieve high-yield stable expression of the target protein in the CHO cells.

[0020] The beneficial effects of the present invention are:

[0021] The CHO cell expression system constructed by the method of the present invention can significantly improve the expression level of the target gene in CHO cells, and achieve transient and stable high-level expression of the target protein in CHO cells. Specifically, the present invention co-transfects the co-transgenic recombinant vector and the target gene recombinant vector into CHO cells to improve the expression level of the target gene, especially in the case where the ratio of the target gene recombinant vector to the co-transgenic recombinant vector is 10:1 and the co-transgenic recombinant vector is 10:1. p18 Gene located in aFGF When the target gene is upstream of the target gene, the expression level of the target gene is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram of the co-transgenic expression vector pIRES-p18 / aFGF;

[0023] Figure 2 Figure 2 is a diagram of the co-transgenic expression vector pIRES-p18-aFGF;

[0024] Figure 3 Figure 2 is a diagram of the co-transgenic expression vector pIRES-aFGF-p18;

[0025] Figure 4 The figure shows the transient expression level of EGFP in CHO cells transfected with different co-transfection ratios (target gene recombinant plasmid: co-transgenic recombinant plasmid);

[0026] Figure 5 This is a graph showing the stable expression level of EGFP in CHO cells transfected with different co-transfection ratios (target gene recombinant plasmid: co-transgenic recombinant plasmid);

[0027] Figure 6 The figure shows the transient and stable expression levels of adalimumab in co-transfected CHO cells;

[0028] Figure 7 The figure shows the transient and stable expression levels of VTN in co-transfected CHO cells. DETAILED DESCRIPTION

[0029] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0030] In the following examples, the CHO cells used were CHO-K1 cells purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0031] The eukaryotic gene expression vector used in the target gene recombinant plasmid is a eukaryotic gene expression vector containing EGFP reporter gene, adalimumab or vitronectin gene constructed on the basis of pIRES-neo vector and is neomycin-resistant.

[0032] The co-transgenic recombinant vector was constructed based on the pIRES-neo vector, and its resistance gene was replaced with blasticidin resistance.

[0033] The recombinant plasmid described in the examples is a recombinant vector.

[0034] Example 1

[0035] The screening of the optimal transfection ratio of the co-transgenic recombinant plasmid and the target gene recombinant plasmid is as follows:

[0036] 1. Transient expression of EGFP at different co-transfection ratios

[0037] 1) Cell culture and transfection

[0038] One day before transfection, select CHO cells in good growth condition and add about 7×10 5 Cells were inoculated into 6-well culture plates. When the cell confluence reached about 80%, plasmid transfection was performed. The plasmids were divided into 5 groups:

[0039] A: Control group: only transfected with the target gene recombinant plasmid;

[0040] Co-rotation group:

[0041] B: target gene recombinant plasmid + co-transgenic recombinant plasmid p18 group;

[0042] C: target gene recombinant plasmid + co-transgenic recombinant plasmid aFGF group;

[0043] D: target gene recombinant plasmid + co-transgenic recombinant plasmid p18 + aFGF group;

[0044] E: target gene recombinant plasmid + co-transgenic recombinant plasmid aFGF+p18 group;

[0045] The target gene recombinant plasmid is pIRES-EGFP, specifically, the EGFP gene is inserted downstream of the CMV promoter of the pIRES-neo vector, and the sequence of the EGFP gene is shown in SEQ ID NO: 3. The co-transgenic recombinant plasmid p18 and the co-transgenic recombinant plasmid aFGF represent recombinant vectors in which p18 or aFGF is inserted downstream of the CMV promoter of the pIRES-neo vector ( Figure 1 ). The co-transgenic recombinant plasmid p18+aFGF and the co-transgenic recombinant plasmid aFGF+p18 represent vectors in which p18 and aFGF are inserted into the pIRES-neo vector, wherein p18+aFGF means that p18 is located downstream of the CMV promoter of the pIRES-neo vector, and aFGF is located downstream of the IRES ( Figure 2); aFGF+p18 means that aFGF is located downstream of the CMV promoter in the pIRES-neo vector, and p18 is located downstream of IRES ( Figure 3 ).

[0046] The steps for plasmid transfection are as follows:

[0047] To each well, add 10 μL of Lipofectamine®2000 transfection reagent to 250 μL of DMEM-F12 serum-free medium to prepare the transfection reagent diluent, mix gently, and place at room temperature for 5 min.

[0048] Add 4 μg of control plasmid to 250 μL DMEM-F12 serum-free medium in each well to prepare control plasmid dilution. For the co-transfection group, add 4 μg of target gene recombinant plasmid and 2 μg, 0.8 μg, 0.4 μg or 0.25 μg of co-transgenic recombinant plasmid to 250 μL DMEM-F12 serum-free medium at a co-transfection ratio of 2:1, 5:1, 10:1, and 15:1, respectively, dilute, and gently mix;

[0049] Will 260 uL of the diluted transfection reagent and After gently mixing the plasmid, incubate at room temperature for 15 min;

[0050] Replace the medium of the cells to be transfected and add 2 mL of fresh culture medium. The obtained transfection reagent dilution and DNA plasmid complex were added to the 6-well plate containing cells to be transfected;

[0051] The transfected cells were cultured in a 37°C, 5% CO2 incubator. Fresh complete medium was replaced 4 to 6 hours after transfection, and suspension culture was performed 48 hours later.

[0052] 2) Cell suspension

[0053] 48 h after transfection, the cells were washed with PBS, the cell pellets were collected by trypsin digestion, and resuspended in CHO serum-free suspension medium. 5 / mL cell density suspension culture 30 mL cell suspension. 37 ℃, CO2 cell culture incubator rotation speed 120 r / min suspension culture, the day of cell inoculation is the suspension culture day 0, the culture continued for 7 days.

[0054] 3) Detection of EGFP transient expression level by flow cytometry

[0055] On the 7th day of cell suspension culture, 1 mL of cells from each group was collected in a centrifuge tube and centrifuged at 1000 rpm for 5 min to collect the cell pellet. The supernatant was discarded, the cell pellet was resuspended in PBS, the cells were washed by pipetting, centrifuged at 1000 rpm for 5 min, and the washing was repeated twice. The supernatant was discarded and the cell pellet was resuspended in 500 μL PBS, and the cell suspension was added to the flow tube for detection.

[0056] The mean fluorescence intensity (MFI) of EGFP in each group of cells was detected by flow cytometry. The MFI of EGFP in the control group was set as 1.

[0057] The results showed that compared with the control group, when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 2:1, the transient expression of EGFP in each group was not significantly improved; when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 5:1, the EGFP expression level in the cells of the co-transfection group was increased, ranging from 1.16 to 1.28 times; when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 10:1, the EGFP expression level in the cells of all co-transfection groups was significantly increased, ranging from 1.48 to 2.35 times; when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 15:1, the EGFP expression level was increased by 1.11 to 1.36 times compared with the control. The results show that when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at different ratios, there were differences in the transient expression level of EGFP in CHO cells, among which the best was when the ratio was 10:1, such as Figure 4 shown.

[0058] 2. Stable expression of EGFP at different co-transfection ratios

[0059] 1) Cell culture and transfection

[0060] The cell culture and transfection were the same as in Example 1.

[0061] 2) Cell stability screening

[0062] 48 hours after transfection, pressure screening was performed using G418 at a final concentration of 800 μg / mL and blasticidin culture medium at 15 μg / mL. The culture medium was replaced every 2 to 3 days. After 7 to 10 days, the cells in the control group died, and the concentration of G418 was reduced to 400 μg / mL, and that of blasticidin was reduced to 10 μg / mL. Culture and screening was continued for 2 to 3 weeks, during which the culture medium was replaced every 2 to 3 days. When stable transformed cell colonies were formed and the cell fusion degree reached 90%, it was a stable cell pool.

[0063] 3) Cell suspension

[0064] The stable selected cells were washed with PBS, the cell precipitates were collected by trypsin digestion, and CHO serum-free medium was added to resuspend the cells. The suspension method was the same as in Example 1.

[0065] 4) Detection of stable expression level of EGFP by flow cytometry

[0066] The flow cytometry detection method is the same as that in Example 1. The results showed that when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 2:1, the expression of EGFP was not significantly increased in each group; when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 5:1, the EGFP expression level in the cells of the co-transfection group was increased, and the increase multiple ranged from 1.27 to 1.65 times; when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 10:1, the EGFP expression level of all co-transfection group cells was increased, and the increase multiple ranged from 2.18 to 3.85 times; when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 15:1, the EGFP expression level was increased by multiples ranging from 1.41 to 2.16 times. The results showed that the stable expression level of EGFP is also optimal when the co-transfection ratio of the target gene recombinant plasmid and the co-transgenic recombinant plasmid is 10:1. Figure 5 shown.

[0067] Example 2

[0068] The effect of co-transgenic on the expression of recombinant protein VTN and adalimumab antibody, the specific steps are as follows:

[0069] 1. Effect of co-transgenesis on transient expression of VTN and adalimumab

[0070] 1) Cell culture and transfection

[0071] One day before transfection, select CHO cells with good growth status and add about 7×10 5 Cells were inoculated into 6-well culture plates and transfected when the cell confluence reached about 80%. Plasmids were divided into 5 groups:

[0072] A: Control group: only transfected with the target gene recombinant plasmid;

[0073] Co-rotation group:

[0074] B: target gene recombinant plasmid + co-transgenic recombinant plasmid p18 group;

[0075] C: target gene recombinant plasmid + co-transgenic recombinant plasmid aFGF group;

[0076] D: target gene recombinant plasmid + co-transgenic recombinant plasmid p18 + aFGF group;

[0077] E: target gene recombinant plasmid + co-transgenic recombinant plasmid aFGF+p18 group;

[0078] The co-transfection group was co-transfected at a ratio of target gene recombinant plasmid: co-transgenic recombinant plasmid = 10:1.

[0079] The target gene recombinant plasmid is a VTN gene or an adalimumab gene inserted downstream of the CMV promoter of the pIRES-neo vector, wherein the VTN gene is shown in SEQ ID NO: 4, the heavy chain of the adalimumab is shown in SEQ ID NO: 5, and the light chain is shown in SEQ ID NO: 6. The co-transgenic recombinant plasmid is the same as the co-transgenic recombinant plasmid of Example 1.

[0080] The transfection steps were the same as those in Example 1. The transfected cells were cultured in a 37°C, 5% CO2 incubator, and fresh complete culture medium was replaced 4 to 6 hours after transfection.

[0081] 2) Cell suspension

[0082] 48 hours after transfection, the cells were cultured in suspension. The experimental steps were the same as those in Example 1. After 7 days of suspension culture, 1 mL of the cell suspension was placed in a centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was taken and centrifuged at 13000 rpm for 20 min. The supernatant was collected for detection.

[0083] 3) ELISA analysis of transient expression of VTN and adalimumab

[0084] Cells were transfected, and the cells were suspended in serum-free water 48 hours after transfection and collected for 7 days. The expressions of adalimumab and VTN were detected by ELISA.

[0085] The results showed that using the CHO cell expression system of the present invention, the transient expression levels of adalimumab and VTN were significantly higher than those of the control group, wherein the transient adalimumab expression levels of the co-transfected p18 and aFGF groups were increased by 1.8 and 1.4 times, respectively, and the co-transfected p18+aFGF and aFGF+p18 groups were increased by 2.9 and 2.2 times, respectively. Figure 6 The transient expression levels of VTN in the co-transfection group of p18 and aFGF increased by 1.6 and 1.2 times, respectively, and the transient expression levels of VTN in the co-transfection group of p18+aFGF or p18+aFGF increased by 2.6 and 2.1 times, respectively. Figure 7 The above results show that the transient expression of the target protein was improved when the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 10:1, and the co-transfection of p18+aFGF plasmid was the most significant.

[0086] 2. Effect of co-transgenic on the stable expression of VTN and adalimumab

[0087] 1) Cell culture and transfection

[0088] The cell culture and transfection steps were the same as in Example 2.

[0089] 2) Cell stability screening

[0090] The experimental steps are the same as in Example 1.

[0091] 3) Cell suspension

[0092] The stable screened cell pool was subjected to suspension culture, and the experimental steps were the same as those in Example 1. After suspension culture for 7 days, 1 mL of the cell suspension was placed in a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was taken, and then centrifuged at 13000 rpm for 20 min, and the supernatant was collected for detection.

[0093] 4) ELISA analysis of stable expression of VTN and adalimumab

[0094] ELISA was used to detect the expression levels of adalimumab and VTN in the collected supernatant. The results showed that compared with the control, the stable expression of adalimumab in the co-transfected p18, aFGF, p18+aFGF and aFGF+ p18 groups increased by 2.62, 2.23, 4.51 and 3.10 times, respectively. Figure 6 As shown, the stable expression of VTN increased by 2.31, 1.84, 4.01, and 3.35 times, respectively. Figure 7 The results were the same as those of transient expression. When the target gene recombinant plasmid and the co-transgenic recombinant plasmid were co-transfected at a ratio of 10:1, the stable expression of the target protein was improved, and the co-transfection of p18+aFGF plasmid was the most significant.

[0095] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention. SEQUENCE LISTING <110> Xinxiang Medical College <120> A method for improving the expression level of recombinant protein in CHO cells <130> none <160> 6 <170> PatentIn version 3.3 <210> 1 <211> 504 <212> DNA <213> Artificial sequence <400> 1 atggccgagc cctggggaa cgagctggcc agcgccgctg ccaggggaga tctggagcag ctgaccagcc tgctgcagaa taatgtgaac gtgaacgccc agaatggctt tggcaggacc gccctgcagg tcatgaagct gggcaatcct gagatcgcca gaagactgct gctgaggggc gccaatcccg atctgaagga taggaccggc ttcgccgtga tccacgacgc cgccagagcc 240 ggatttctgg acacactgca gacactgctg gagtttcagg ccgacgtgaa catcgaggat aacgagggca atctgcccct gcacctggcc gccaaggagg gacacctgag ggtggtggag 360 ttcctggtga agcacatggc ctccaacgtg ggccacagaa accacaaggg cgataccgcc 420 tgtgatctgg ccagactgta cggcaggaat gaggtggtga gcctgatgca ggccaacggc 480 gccggcggag ctaccaatct gcag 504 <210> 2 <211> 465 <212> DNA <213> The snowstorm <400> 2 atggccgagg gcgagatcac aacattcact gccctgacag agaagtttaa tctgcctcct 120. ggcaactaca agaagcctaa gctgctgtac tgctccaatg gcggccactt cctgaggatc ttgcctgacg gcaccgtgga tggcacaagg gataggagcg atcagcacat ccagctgcag 180 ctgagcgccg agtccgtggg cgaggtgtac atcaagagca ccgagacagg ccagtacctg 240 gccatggaca ccgatggcct gctgtacggc agccagacac ctaatgagga gtgcctgttc 300 ctggagagac tggaggagaa ccactacaac acctacatca gcaagaagca cgccgagaag 360 aactggtttg tgggcctgaa gaagaacggc tcctgtaaga ggggccccag gacccactac 420 ggccagaagg ccatcctgtt tctgcccctg cctgtgagca gcgat 465 <210> 3 <211> 720 <212> DNA <213> Artificial Sequence <400> 3 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga 720 <210> 4 <211> 1443 <212> DNA <213> Artificial Sequence <400> 4 atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatatcc 60 agaggagacc aggagtcctg caagggcaga tgtacagagg gctttaacgt ggataagaag 120 tgccagtgcg acgagctgtg ctcctactac cagtcctgct gcacagacta cacagccgag 180 tgtaagcccc aggtgacaag aggcgacgtg ttcaccatgc ccgaggacga gtacacagtg 240 tacgacgacg gcgaggagaa gaacaacgcc accgtgcacg agcaggtggg cggccctagc 300 ctgacctccg atctgcaggc ccagagcaag ggcaaccctg agcagacccc cgtgctgaag 360 cctgaggagg aggcccctgc ccccgaggtt ggagcttcca agcccgaggg catcgattcc 420 agacctgaga ccctgcaccc cggcaggcct cagcctccag ctgaggagga gctgtgcagc 480 ggcaagccct tcgacgcctt tacagacctg aagaacggct ccctgttcgc ctttagaggc 540 cagtactgtt acgagctgga tgagaaggcc gtgaggcctg gctaccctaa gctgatcaga 600 gatgtgtggg gcatcgaggg ccccatcgac gccgctttta ccagaatcaa ttgtcagggc 660 aagacctacc tgtttaaggg ctcccagtac tggaggtttg aggacggcgt gctggaccct 720 gactacccta gaaacatctc cgatggcttc gacggcatcc ctgataacgt ggacgccgcc 780 ctggccctgc ccgctcacag ctattccggc agggagagag tgtacttctt taagggcaag 840 cagtactggg agtaccagtt ccagcaccag cccagccagg aggagtgcga gggcagcagc 900 ctgtccgccg tgtttgagca cttcgccatg atgcagaggg atagctggga ggatatcttc 960 gagctgctgt tctggggcag aacctccgcc ggcaccagac agcctcagtt tatctccaga 1020 gactggcacg gcgtgcccgg ccaggtggat gctgctatgg ccggcagaat ctacatctcc 1080 ggcatggccc ctaggccttc cctggccaag aagcagaggt tcagacacag gaacagaaag ggctacaggt cccagagagg ccactccagg ggcagaaacc agaatagcag aagaccttcc agggccacct ggctgagcct gttctccagc gaggagagca atctgggcgc caacaactac gatgattaca gatggattg gctggtgccc gccacctgcg agcccatcca gtccgtgttc 1320. ttcttctccg gcgataagta ctacagggtg aacctgagga caaggagagt ggataccgtg 1380. 1440. gaccctccct accccagaag catcgccccag tactggctgg gctgtcccgc ccctggccac ctg 1443 <210> 5 <211> 1356 <212> DNA <213> The snowstorm <400> 5 gaagtgcaat tggttgaatc aggagga ctcgtgcaac cggaggag tttacgatta tcttgtgctg cctctggatt cacctttgac gactatgcaa tgcattgggt ccgtcaagca ccaggaaaag gtttagagtg ggtttcagca atcacttgga actccggaca tattgactat 180 gccgatagtg ttgagggtcg attcacaatc tcacgagata acgcgaagaa tagtctatac 240 ctacagatga atagcctaag agctgaggat actgccgttt attactgtgc aaaggtttcc 300 tatctttcta ctgcatctag tcttgattac tggggacaag gaacacttgt cacagtttcc 360 tctgctagca caaaaggacc tagcgttttc cctctggcac catcaagtaa gagcaccagt 420 ggcgggacag cagcactggg ttgtcttgtg aaagactatt tcccagaacc cgttaccgtt 480 agttggaact caggcgcact tacttcggga gttcatactt ttcctgctgt cttacaatct 540 tccggtctct attcactaag ctcagttgtc actgtacctt cctcaagcct tgggacacaa 600 acctacattt gtaacgtcaa tcataaaccg agcaatacga aggtagataa gaaagtcgag 660 ccaaagagtt gtgataaaac acacacttgc ccaccttgcc cagctcctga actcttaggt 720 ggaccaagcg ttttcctctt tcctccaaag ccgaaagata cacttatgat atcacgcaca 780 cccgaagtta cttgtgtggt tgtagacgtt tctcatgaag atcccgaagt gaagtttaat 840 tggtacgtcg atggtgttga agttcacaat gctaagacta agccaagaga agagcaatac 900 aactcaacct atagagttgt ttccgtctta accgtactgc atcaagattg gttgaacggc 960 aaggagtata aatgcaaggt tagcaataaa gcactacctg caccgattga gaagacaatt 1020 agcaaagcaa aaggacaacc aagggaacca caagtctata cacttccacc ttcaagggat 1080 gagctgacta agaatcaagt atccttgacc tgtttagtca aggggtttta cccttctgac 1140 attgccgtag aatgggaatc taatgggcag cctgagaata actataagac aactccaccc 1200 gtactcgatt ctgacggctc ttttttccta tactccaagc taaccgtgga taaatcacgt 1260 tggcaacaag gaaacgtttt ctcttgttct gtgatgcacg aggctttgca taatcactac 1320 acacaaaaga gcttaagtct tagccctggg aaatag 1356 <210> 6 <211> 645 <212> DNA <213> Artificial Sequence <400> 6 gacatacaaa tgactcaatc tccaagttca ctatctgctt cagtcggcga tagggtcact 60 ataacttgta gagcctctca gggcataaga aactatttgg catggtacca acagaaacct 120 ggaaaagctc ctaagctgct aatatatgct gcttctacac ttcagagtgg agtaccttca 180 agattcagtg gatctggttc tgggactgat ttcactttga ctatctcatc cctccaacca 240 gaagacgttg ctacatacta ttgccagcgc tataataggg ctccttatac ctttggacaa 300 ggcacaaaag tcgagattaa gagaactgtt gctgcaccat cagtgtttat tttccctcca 360 420 cctagagaag cgaaagtcca atggaaagta gataacgcac ttcagtctgg gaactcacaa 480 gagagtgtca ctgaaaga ttcgaaagac tctacctatt cactctcatc gactcttact 540 ctgtcaaaag ctgattacga gaagcaaa gtgtatgctt gcgaagttac acaccaagga 600 cttagctcac cagataaccaa gagcttcaat aggggagaat gctga 645

Claims

1. A method for increasing the expression level of a recombinant protein in CHO cells, characterized in that: The following steps are involved: (1) Combine the target gene recombinant vector with the cell cycle regulation-related gene p18 and genes that regulate cell growth aFGF The co-transgenic recombinant vector is constructed based on the pIRES-neo vector and contains p18 + aFGF eukaryotic expression vector; p18 Gene located in aFGF Upstream of the gene; The target gene recombinant vector is a eukaryotic expression vector containing an EGFP reporter gene, an adalimumab gene or a vitronectin gene; The gene p18 The sequence is shown in SEQ ID NO: 1; The gene aFGF The sequence is shown in SEQ ID NO: 2; (2) After co-transfection of the co-transgenic recombinant vector and the target gene recombinant vector into CHO cells, the cells are cultured and screened to obtain a CHO cell expression system; the co-transfection ratio of the target gene recombinant vector and the co-transgenic recombinant vector is 10:1; (3) Transiently and stably transfected CHO cell lines were cultured in suspension without serum to obtain the target protein.

2. The method for improving the expression level of recombinant protein in CHO cells according to claim 1, characterized in that: The CHO cell is any one of CHO-K1, CHO-S, and DG44.

3. The method for improving the expression level of recombinant protein in CHO cells according to claim 1, characterized in that: The transfection method is calcium phosphate method, electroporation, liposome transfection or PEI transfection.

4. A CHO cell expression system constructed using the method according to any one of claims 1 to 3.

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