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WO2020088302A1 - Utilisation de nw_006882456-1 dans le génome de cellule cho dans l'expression stable de protéine - Google Patents

Utilisation de nw_006882456-1 dans le génome de cellule cho dans l'expression stable de protéine Download PDF

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Publication number
WO2020088302A1
WO2020088302A1 PCT/CN2019/112454 CN2019112454W WO2020088302A1 WO 2020088302 A1 WO2020088302 A1 WO 2020088302A1 CN 2019112454 W CN2019112454 W CN 2019112454W WO 2020088302 A1 WO2020088302 A1 WO 2020088302A1
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target sequence
use according
protein
site
cho cell
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Chinese (zh)
Inventor
李华钟
金坚
周松涛
陈蕴
段作营
龚笑海
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to the field of gene technology, and in particular to a recombinant gene of CHO cells for stable protein expression.
  • Chinese hamster ovary cells Choinese Hamster Ovary cells, CHO
  • CHO Chinese hamster Ovary cells
  • the increase in the number of transgenic copies and the increase in the yield of the target protein is not a clear positive correlation.
  • the expression level of most CHO cells is unstable.
  • the currently widely used methods for constructing stable transfected cells are time-consuming and labor-intensive, mainly because of the need to repeat a large number of monoclonal screening processes, so the current field of cell line construction is generally expected to develop a method that can achieve high
  • the method of expressing and stably expressing cells and can ensure that the constructed recombinant cell line has the same quality level as the traditional method to ensure the approval of the regulatory agency.
  • exogenous protein-expressing cell lines The traditional method for constructing exogenous protein-expressing cell lines is to randomly integrate exogenous genes into the cell genome, and then screen through a series of high-expression monoclonal cells to obtain exogenous protein high-expression cell lines. Due to the diversity of site effect differences, the expression levels of recombinant cells produced by random integration are different, so it takes a long time and many steps in the later stage to select high-expression monoclonal cells. Monoclonal cells obtained through random integration cannot guarantee the stable expression of peptides / proteins during cell passage, and each recombination cell construction requires repeated monoclonal screening, increasing the research and development costs of biopharmaceuticals.
  • the applicant of the present invention provides an application in which NW_006882456-1 stably expresses proteins in the CHO cell genome.
  • the application of the present invention introduces different protein genes at a fixed position in the CHO cell genome and performs stable expression; in addition, in the process of realizing this fixed-point integration, there is no need to select monoclonals multiple times to obtain higher expression cell lines, saving a lot time.
  • An application for stably expressing a protein at a site in the CHO cell genome is located at base 1020651 of the CHO cell gene NW_006882456.1;
  • the protein is a protein with a molecular weight of less than 160 KDa.
  • the protein is one of a polypeptide, a functional protein, an antibody, and a fusion protein.
  • target sequence is 5'-ACCCTTGTGCCCCAAAGACAGGG-3 '.
  • the target sequence is 5'-ACCCTGTCTTTGGGGCACAAGGG-3 '.
  • the target sequence is 5'-GTCTTTGGGGCACAAGGGTGAGG-3 '.
  • the target sequence is 5'-TTTGGGGCACAAGGGTGAGGTGG-3 '.
  • the target sequence is 5'-CAAGGGTGAGGTGGGGTAAATGG-3 '.
  • the target sequence is 5'-GGTGAGGTGGGGTAAATGGATGG-3 '.
  • the target sequence is 5'-AGGTGGGGTAAATGGATGGTTGG-3 '.
  • the target sequence is 5'-GGGGTAAATGGATGGTTGGTTGG-3 '.
  • the target sequence is 5'-TGGGGCACAAGGGTGAGGTGGGG-3 '.
  • the target sequence is 5'-ACAGAGAAACCCTGTCTTTGGGG-3 '.
  • the target sequence is 5'-CACAGAGAAACCCTGTCTTTGGG-3 '.
  • the target sequence is 5'-AGTGAGTTCTAAGACAGCCAGGG-3 '.
  • the target sequence is 5'-ACACAGAGAAACCCTGTCTTTGG-3 '.
  • the target sequence is 5'-GTTTCTCTGTGTAACAGCCCTGG-3 '.
  • the target sequence is 5'-ACTCACTCTGTAGACCAGGCTGG-3 '.
  • a targeting vector containing the 5'NNNNNNNNNNNNNNNNNNNNNNNGNG3 'target sequence that can be used for site editing and can be recognized by CRISPR / Cas9 technology.
  • the preparation method of the targeting vector is as follows: the PSK-u6-gRNA plasmid is digested with BbsI, and the target sequence DNA fragment containing the sticky end of BbsI is connected to obtain the targeting vector.
  • a recombinant donor vector for expressing foreign genes in CHO cells the recombinant donor vector containing 500bp-800bp homology arms at the 1020651 base of the site NW_006882456.1.
  • the preparation method of the recombinant donor vector is as follows: the protein gene is inserted into the region between the 5'arm and the 3'arm of the plasmid, so that the nucleotide sequence is located downstream of the promoter and regulated by it, and is expressed by recombinant CHO cells Plasmid.
  • the promoters are: CMV (human cytomegalovirus-derived strong mammalian expression promoter), EF-1a (human elongation factor 1 ⁇ -derived strong mammalian expression promoter), SV40 (simian vesicular virus 40-derived lactation) Animal expression promoter), PGK1 (a mammalian promoter derived from the phosphoglycerate kinase gene), UBC (a mammalian promoter derived from the human ubiquitin C gene), human beta-actin (a mammal derived from the ⁇ -actin gene) Animal promoter), CAG (strong hybrid mammalian promoter).
  • a CHO recombinant cell line for stably expressing foreign proteins uses the vector or plasmid during the construction process.
  • a method for CHO cell gene expression protein characterized in that it includes the following steps:
  • Cas9, the targeting vector and the recombinant donor vector are used to transform CHO cells together to obtain recombinant CHO cells;
  • Recombinant CHO cells are cultured on the plate, collect the supernatant to detect the expression level, and suspend the acclimated recombinant CHO cells;
  • the present application also provides a selection of stable expression sites in the CHO cell genome:
  • the PCR products are electrophoresed, and the main band is cut and recovered for sequencing.
  • select the relevant information of the CHO cell line with only a single copy of lentivirus integration and compare its sequence information with the CHO-K1 genome information on BLAST to find high expression Integration site.
  • the original site information was changed from the 1020651 base at the CHO cell gene NW_006882456.1 to the 148091 base at the CHO cell gene NW_003614241.1.
  • the range was changed from 1020585-1020690 to 148052-148157.
  • the invention adopts a fixed-point integration method.
  • By integrating the targeted gene into a stable expression region it can well overcome the problem of integration site uncertainty caused by random integration, and effectively avoids repeated multiple rounds of high-expression monoclonal screening , Can effectively reduce the research and development time of biopharmaceutical construction of stable expression cell lines, and reduce costs.
  • protein genes are introduced at a fixed position of CHO cell genes and are stably expressed.
  • Figure 1 is a schematic diagram of the present invention
  • Figure 2 shows the results of gene identification of CHO cells introduced with NGGH 75KDa gene
  • Figure 3 is the sequencing of CHO cell genes introduced with NGGH 75KDa gene by OoPCR_fwd and OoPCR_rev;
  • Figure 6 shows the quality of antibody protein secreted by each recombinant CHO cell daily.
  • FIG. 1 is a schematic diagram of a donor plasmid for integration into the site and a schematic diagram of how to integrate the site into the site by homologous recombination.
  • GOI is our target gene. It uses 5'arm and 3'arm homologous recombination arms under the pressure of 4 ⁇ g / mL puromycin screening, and integrates into the target site in a targeted manner.
  • sequence upstream of the 5’arm is a negative selection tag, which can be used to remove randomly integrated monoclonal cells to ensure that the resulting recombinant CHO cell line is site-directed integration.
  • NW_006882456.1 NW_003614241.1 (chromosome 6), 1020651 (148091) base is integrated, and the Zsgreen1 gene is integrated there.
  • the fluorescent cells were subcultured for at least 50 generations, and the expression level of fluorescence was detected by flow cytometry. The 50th generation of fluorescent cells still have a good green fluorescent protein expression level, and the fluorescent signal can be stably retained during the passage of cells.
  • this fluorescent cell was also subjected to suspension acclimation, and the expression level of the fluorescent protein after suspension acclimation was again detected by flow cytometry.
  • the test results show that more than 95% of the recombinant CHO cells suspended for 50 passages still maintain the expression of green fluorescent protein after suspension. It can be considered that the site is extremely stable and will not lose site integration due to cell passage. Fluorescent protein gene.
  • the parameter settings are as follows: 1) The maximum number of mismatched bases at the beginning 15 bp after NGG is 0; 2) The number of mismatched bases at 21 bp after NGG is 2.
  • CMV human cytomegalovirus-derived strong mammalian expression promoter
  • CMV human cytomegalovirus-derived strong mammalian expression promoter
  • EF-1a human elongation factor 1 ⁇ -derived strong mammalian expression promoter
  • SV40 synthetic Mammalian expression promoter derived from vacuolar virus 40
  • PGK1 a mammalian promoter derived from phosphoglycerate kinase gene
  • UBC a mammalian promoter derived from human ubiquitin C gene
  • human beta actin ⁇ - Common promoters such as mammalian promoters derived from actin genes
  • CAG strong hybrid mammalian promoters
  • HSA human serum albumin gene
  • sgRNA-1rev 5 TAAAACTGTCTTTGGGGCACAAGGGT 3’
  • PSK-u6-gRNA plasmid is digested with BBsI, and the cut vector is recovered;
  • the M13-synthesized R primer can amplify the band as a positive clone.
  • donor plasmaid The specific information of donor plasmaid is shown in Figure 1: The rest are all synthesized except GOI; the 600bp sequence upstream and downstream of the target is the sequence information of the left and right homology arms of donor plasma, and GOI is the HSA gene that passes the existing Novizan company
  • the C115 kit completes the process of integrating HSA into donor plasma.
  • Co-transfect Cas9 (donated by Dr. Helene F Kildegaard, Danish University of Science and Technology), SgRNA and Donor plasmid to CHO cells cultured at 37 °C and 5% CO 2 at a molar ratio of 1: 1: 1.
  • the transfection reagent is Lipofectamine 3000 (Thermo Fisher Scientific), the specific transfection method refers to the instruction manual, and then the cells are added with 4 ⁇ g / mL puromycin for screening, and the process takes 10 days in total; then the MoFloXDP FACS machine (Beckman Coulter) is used for monoclonal cell separation Select, select cells that do not contain any fluorescence, and seed them into 96-well plates.
  • the specific information of the donor plasmaid is shown in Figure 1: Except GOI, the rest are synthetic; the 600bp sequence upstream and downstream of the target is the sequence information of the left and right homology arms of the donor plasma, GOI is the NGGH gene passed the existing Novizan company
  • the C115 kit completes the process of integrating NGGH onto the donor plasmad.
  • sgRNA and DonorPlasmid need to be constructed as follows:
  • the sgRNA construction is the same as in Example 4.
  • the specific information of the donor plasmaid is shown in Figure 1: Except for GOI, the rest are synthetic; the 600bp sequence upstream and downstream of the target is the sequence information of the left and right homology arms of the donor plasma, and GOI is the Avastin gene through the existing Novizan company
  • the C115 kit completes the process of antibody gene integration into donor plasma.
  • Cas9 (donated by Dr. Helene F Kildegaard, Danish University of Science and Technology), SgRNA and Donor plasmid were co-transfected into CHO cells cultured at 37 °C, 5% and CO 2 with a molar ratio of three plasmids of 1: 1 : 1, the transfection reagent is Lipofectamine 3000 (Thermo Fisher Scientific), please refer to the instruction manual for the specific transfection method. After that, cells were added with 4 ⁇ g / mL puromycin for screening, and the process totaled 10 days. Then use MoFloXDP FACS machine (Beckman Coulter) for monoclonal cell sorting, select cells that do not contain any fluorescence, and seed them into 96-well plates.
  • Figure 2 shows the identification result of the CHO gene introduced with the 75KDa gene of NGGH.
  • Lanes 1-3 are the 5'jucntion PCR results of three monoclonal cells, and lanes 4-5 are the 3'junction PCR results. There are significant bands. Gene knock-in.
  • Figure 3 uses OoPCR_fwd and OoPCR_rev for sequencing to determine the sequence accuracy at the junction (5'junction and the junction of the 5 'upstream of the 3'junction and the genome). The sequencing results verify that the GOI is accurately inserted into the target location area .
  • the three cell lines prepared in Examples 4-6 were subjected to ELISA test to observe whether the target protein was expressed and whether it was stable long-term expression.
  • Detection method The detection of the four is carried out by ELISA method. All the positive cells selected in the 6-well plate are cultured to check whether they have long-term stable expression of the target protein.
  • the kit used is Abbclonal Human Albumin ELISA Kit ( RK00157) and Human IgG (Total) ELISA Kit (RK00393).
  • Figures 4 and 5 show the cell expression of HSA and NGGH under different passages, and the ordinate is the amount of secreted protein per cell per day.
  • the NGGH and HSA can stably express the corresponding genes within 50 generations in the plate, and the target protein expression levels of the three selected NGGH site-integrated cell lines and two HSA site-integrated cell lines tend to be close.
  • Figure 6 shows the mass of antibody protein secreted by each recombinant CHO cell daily. Obviously, the cells can stably express and secrete the corresponding protein under different passage conditions, which shows a good stability, which is consistent with the previous fluorescent cell results. The results show that the site can perform CRISPR / Cas9-mediated site-directed integration, and can stably express the corresponding protein.
  • the target sequences described in claims 5-19 can successfully construct a fixed-point integration and stable expression cell line, and all can be stably expressed Target protein.

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Abstract

La présente invention concerne l'utilisation de NW_006882456-1 dans un génome de cellule CHO dans l'expression stable de protéine, le site d'expression stable de protéine dans le génome de cellule CHO étant localisé à la position de base 1020651 du gène NW_006882456.1 dans la cellule CHO. 5'NNNNNNNNNNNNNNNNNNNNNGG3', qui est près des sites s'étendant de 1020585-1020690 et qui est identifié par la technologie CRISPR/Cas9, est la séquence cible. Différents gènes de protéine sont introduits à une position fixe du génome de cellule CHO et l'expression stable est effectuée.
PCT/CN2019/112454 2018-10-30 2019-10-22 Utilisation de nw_006882456-1 dans le génome de cellule cho dans l'expression stable de protéine Ceased WO2020088302A1 (fr)

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CN109295093B (zh) * 2018-10-30 2021-08-03 江南大学 一种cho细胞基因组内nw_006882456-1稳定表达蛋白质的应用
CN113969283B (zh) * 2021-11-23 2022-07-12 江南大学 一种cho细胞基因nw_003613756.1内稳定表达蛋白质的位点及其应用
CN113969284B (zh) * 2021-11-23 2022-07-12 江南大学 一种cho细胞基因nw_003614889.1内稳定表达蛋白质的位点及其应用

Citations (5)

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CN106554973A (zh) * 2015-09-30 2017-04-05 北京吉尚立德生物科技有限公司 一种cho细胞分泌能力评价系统
WO2017186671A1 (fr) * 2016-04-25 2017-11-02 Danmarks Tekniske Universitet Cellules mammifères modifiées pour la production de protéines recombinantes
CN107557390A (zh) * 2017-09-18 2018-01-09 江南大学 一种筛选cho细胞系高表达位点的方法
WO2018148196A1 (fr) * 2017-02-07 2018-08-16 Sigma-Aldrich Co. Llc Intégration ciblée stable
CN109295093A (zh) * 2018-10-30 2019-02-01 江南大学 一种cho细胞基因组内nw_006882456-1稳定表达蛋白质的应用

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EP3209785B1 (fr) * 2014-10-23 2019-07-31 Regeneron Pharmaceuticals, Inc. Nouveaux sites d'intégration cho et leurs utilisations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106554973A (zh) * 2015-09-30 2017-04-05 北京吉尚立德生物科技有限公司 一种cho细胞分泌能力评价系统
WO2017186671A1 (fr) * 2016-04-25 2017-11-02 Danmarks Tekniske Universitet Cellules mammifères modifiées pour la production de protéines recombinantes
WO2018148196A1 (fr) * 2017-02-07 2018-08-16 Sigma-Aldrich Co. Llc Intégration ciblée stable
CN107557390A (zh) * 2017-09-18 2018-01-09 江南大学 一种筛选cho细胞系高表达位点的方法
CN109295093A (zh) * 2018-10-30 2019-02-01 江南大学 一种cho细胞基因组内nw_006882456-1稳定表达蛋白质的应用

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