WO2023131330A1 - Expression vector and use thereof - Google Patents

Expression vector and use thereof Download PDF

Info

Publication number
WO2023131330A1
WO2023131330A1 PCT/CN2023/071371 CN2023071371W WO2023131330A1 WO 2023131330 A1 WO2023131330 A1 WO 2023131330A1 CN 2023071371 W CN2023071371 W CN 2023071371W WO 2023131330 A1 WO2023131330 A1 WO 2023131330A1
Authority
WO
WIPO (PCT)
Prior art keywords
expression vector
screening
expression
marker
host cells
Prior art date
Application number
PCT/CN2023/071371
Other languages
French (fr)
Chinese (zh)
Inventor
沈潇
李京浩
弗兰克·亚娜
Original Assignee
佛山汉腾生物科技有限公司
广州汉腾生物科技有限公司
佛山普津生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 佛山汉腾生物科技有限公司, 广州汉腾生物科技有限公司, 佛山普津生物技术有限公司 filed Critical 佛山汉腾生物科技有限公司
Publication of WO2023131330A1 publication Critical patent/WO2023131330A1/en

Links

Images

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
    • C12N9/003—Dihydrofolate reductase [DHFR] (1.5.1.3)
    • 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
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
    • C12Y105/01003—Dihydrofolate reductase (1.5.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
    • C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/36—Vector systems having a special element relevant for transcription being a transcription termination element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to an expression vector and a method for screening cell pools using it.
  • CHO cells Chinese hamster ovary (CHO) cells are the most commonly used expression hosts for the manufacture of complex biomolecules.
  • the two most common methods for expressing proteins in CHO cells are transient gene expression and stable gene expression.
  • Transient gene expression means that after the exogenous gene enters the recipient cell, it exists on the free carrier and does not integrate into the chromosome of the cell.
  • Transient gene expression is the method of choice for rapid production of small quantities of recombinant protein, usually in as little as 1-3 weeks.
  • transient gene expression is not practical when gram-scale protein production is required due to the large amounts of DNA and host cells required. Therefore, stable gene expression is often used to produce gram-scale recombinant proteins.
  • Stable gene expression refers to the integration of the transfected target gene into the chromosomal DNA to express the target protein.
  • stabilizing gene expression is time-consuming and laborious. It usually takes 3-9 months to generate CHO cell lines. Therefore, there is an urgent need for cell lines that are fast, stable, and high in producing target proteins.
  • the invention provides an expression vector, through which a cell pool can be rapidly screened, and a CHO clone can be obtained through further screening, so as to generate a stable CHO cell line.
  • a cell pool can be rapidly screened, and a CHO clone can be obtained through further screening, so as to generate a stable CHO cell line.
  • the expression level of the target protein is increased, and the rapidly produced CHO cell line is not only stable but also highly productive.
  • the present invention provides an expression vector, which includes a selection marker expression unit, at least one target gene expression unit, and inverted terminal repeats (ITRs) of the PiggyBac transposon.
  • ITRs inverted terminal repeats
  • the screening marker expression unit comprises a coding sequence of a screening marker, and a first regulatory sequence operably linked to the coding sequence of the screening marker, for example, a coding sequence of a promoter and a coding sequence of a terminator.
  • the selectable markers include, but are not limited to, Puromycin (Puro), Neomycin (Neo), Hygromycin B (Hygro B), and Blasticidin ( Blasticidin, Bsd), glutamine synthetase (Glutamine synthetase, GS) and dihydrofolate reductase (Dihydrofolate reductase, DHFR).
  • the screening marker is human GS.
  • the promoter of the expression unit of the selection marker can be a herpes simplex virus thymidine kinase (Thymidine kinasegene of Herpes simplex virus, HSV-tk) promoter or a simian vacuolar virus 40 (Simian virus40, SV40) promoter son.
  • the terminator of the selection marker expression unit can be a polyadenylation signal (PolyA), such as SV40 PolyA, bovine growth hormone (Bovine growth hormone, bGH) PolyA, HSV-tk PolyA or rabbit ⁇ - Globin (Rabbit beta-globin, RBG) PolyA, preferably SV40 PolyA.
  • PolyA polyadenylation signal
  • SV40 PolyA bovine growth hormone (Bovine growth hormone, bGH) PolyA
  • HSV-tk PolyA HSV-tk PolyA or rabbit ⁇ - Globin (Rabbit beta-globin, RBG) PolyA, preferably SV40 PolyA.
  • the screening marker expression unit comprises the coding sequence of HSV-tk promoter and the coding sequence of SV40 polyA.
  • the screening marker expression unit comprises the coding sequence of the HSV-tk promoter, the coding sequence of the human GS screening marker and the coding sequence of SV40 polyA.
  • the target gene expression unit comprises the target gene sequence and a second regulatory sequence operably linked to the target gene sequence, for example, the coding sequence of the target gene expression regulatory element and the coding sequence of the terminator.
  • the coding sequence regulatory sequence of the target gene expression regulatory element has a sequence selected from the sequences shown in SEQ ID NO.1-6.
  • the terminator of the target gene expression unit can be polyA, such as SV40 PolyA, bGH PolyA, tk PolyA, RBG PolyA, preferably bGH PolyA.
  • the target gene is one or more, such as 2, 3 or 4, and correspondingly, the expression vector has 2, 3 or 4 target gene expression units.
  • the target gene encodes the light chain and/or heavy chain of an anti-CD20 monoclonal antibody (Rituximab) or an anti-HER2 monoclonal antibody (Trastuzumab).
  • the expression vector has 1 or 2 target gene expression units.
  • the expression vector can be quickly screened to obtain a CHO cell pool, and further screened to obtain a CHO clone to generate a stable CHO cell line, and by optimizing the regulatory elements in the expression vector, the expression of the target protein is increased, and the rapid production
  • the CHO cell line is not only stable but also highly productive.
  • the present invention provides an expression system comprising the expression vector of the first aspect.
  • the expression system further comprises a helper plasmid encoding a PiggyBac transposase.
  • the present invention provides host cells transfected with the expression vector of the first aspect.
  • the host cell is transfected with the expression system of the second aspect.
  • the expression vector of the first aspect is stably integrated in the genome of the host cell.
  • the host cell does not contain a helper plasmid encoding a PiggyBac transposase.
  • the host cell is a CHO cell, such as a CHO-K1 cell or a CHOExpress TM cell.
  • the host cell is a stable CHO cell line.
  • the host cell has increased expression of the protein of interest.
  • the present invention provides a method for screening cells using the expression vector of the first aspect or the expression system of the second aspect, the method comprising the following steps:
  • step (b) a step of screening the host cells obtained in step (a) with a screening reagent directed against the screening marker in the expression vector.
  • the host cell is a CHO cell, such as a CHO-K1 cell or a CHOExpress TM cell.
  • the selection marker is puromycin, and 10 ⁇ g/ml of puromycin is used to select host cells transfected with the expression vector comprising a puromycin resistance selection marker .
  • the selection marker is puromycin, and increased concentrations of puromycin (10 ⁇ g/ml, 50 ⁇ g/ml, 250 ⁇ g/ml) are used to select host cells transfected with The expression vector for the puromycin resistance selection marker.
  • the selection marker is GS, and 25 ⁇ M methionine sulfoximine (MSX) is used to select host cells transfected with the expression vector comprising the GS selection marker .
  • MSX methionine sulfoximine
  • the selection marker is GS, and an increased concentration of MSX (25 ⁇ M, 50 ⁇ M) is used to select host cells transfected with the expression vector comprising the GS selection marker.
  • the selection marker is GS, and increased concentrations of MSX (25 ⁇ M, 50 ⁇ M, 100 ⁇ M) are used to select host cells transfected with the expression vector comprising the GS selection marker.
  • the screening marker is human GS.
  • the selection marker is DHFR, and 250 nM of methotrexate (methopterin, MTX) is used to select host cells transfected with the expression vector comprising the DHFR selection marker.
  • methotrexate methopterin, MTX
  • the selection marker is DHFR
  • increased concentrations of methotrexate 250 nM, 500 nM are used to select host cells transfected with the expression vector comprising the DHFR selection marker.
  • the screening reagent is puromycin, methionine iminosulfone or methotrexate.
  • the host cells are subjected to pressure selection with CHO cell culture medium containing puromycin, methionine sulfone imino, or methotrexate.
  • the medium is BalanCD CHO GROWTH A medium.
  • the screening pressure is removed after the viability rate recovers to above 90%.
  • the screening method of the present invention screens high-yield and stable CHO clones, and then produces stable CHO cell lines.
  • the present invention provides cells obtained by the screening method of the fourth aspect.
  • the present invention provides the expression vector of the first aspect, the expression system of the second aspect, the host cell of the third aspect, or the screening method of the fourth aspect, or the purpose of increasing the expression level of the cells of the fifth aspect protein application.
  • the expression vector provided in the present invention can be quickly screened to obtain a cell pool through the expression vector, and can be further screened to obtain a CHO clone to produce a stable CHO cell line, which greatly shortens the time for producing a stable CHO cell line.
  • the optimization of the regulatory elements in the medium increases the expression of the target protein, and the rapidly produced CHO cell line is not only stable but also highly productive.
  • Figure 1 shows a structural diagram of an expression vector comprising two target gene expression units.
  • Figure 2 shows the structural diagram of an expression vector comprising an expression unit of a target gene.
  • Figure 3 shows the selection strategy against the pool of cells using the puromycin resistance selection marker.
  • Figure 4 shows the screening strategy for cell pools using GS selection markers including rat GS as well as human GS.
  • Figure 5 shows the screening strategy against the cell pool using the DHFR selection marker.
  • Figure 6 shows the expression vector structure for expressing anti-CD20 mAb (Rituximab).
  • Figure 7 shows the cell viability (VIA) of the CHO-K1 cell pool.
  • Figure 8 shows the cell viability (VIA) of the CHOExpress TM cell pool.
  • Figure 9 shows the viable cell density (VCD) and cell viability (VIA) of the CHO-K1 cell pool with better recovery of cell viability.
  • Figure 10 shows the viable cell density (VCD) and cell viability (VIA) of the CHOExpress TM cell pool with better recovery of cell viability.
  • Figure 11 shows the growth curve of the cell pool at 37°C, where "KP10” refers to K1-PR10, “KP250” refers to K1-PR250, "EP10” refers to EXP-PR10, “EP250” refers to EXP-PR250, "KH25 “ refers to K1-HG25, “KH50” refers to K1-HG50, “KH100” refers to K1-HG100, “EH25” refers to EXP-HG25, “EH50” refers to EXP-HG50, and “EH100” refers to EXP-HG100.
  • Figure 12 shows the growth curve of the cell pool when the temperature was lowered to 33°C on the sixth day, where "KP10” refers to K1-PR10, “KP250” refers to K1-PR250, “EP10” refers to EXP-PR10, and “EP250” refers to EXP -PR250, "KH25” refers to K1-HG25, “KH50” refers to K1-HG50, “KH100” refers to K1-HG100, “EH25” refers to EXP-HG25, “EH50” refers to EXP-HG50, “EH100” refers to EXP-HG100 .
  • Figure 13 shows the results of Protein A HPLC detection expression level, where "KP10” refers to K1-PR10, “KP250” refers to K1-PR250, “EP10” refers to EXP-PR10, “EP250” refers to EXP-PR250, "KH25” refers to K1-HG25, “KH50” refers to K1-HG50, “KH100” refers to K1-HG100, “EH25” refers to EXP-HG25, “EH50” refers to EXP-HG50, and “EH100” refers to EXP-HG100.
  • Figure 14 shows the growth curve of the cells in Example 3.
  • FIG. 15 shows the expression detection results in Example 3.
  • FIG. 16 shows the expression detection results in Example 4.
  • the present invention can construct an expression vector comprising two target gene expression units (see FIG. 1 ), or construct an expression vector comprising one target gene expression unit (see FIG. 2 ).
  • the PiggyBac system that the present invention adopts comprises two vectors, and a vector is called helper plasmid, is responsible for coding transposase; Another vector is called transposon plasmid, comprises two terminal repeats (ITRs) and between the two The transposed region, promoter, expression cassette and gene of interest are cloned into this transposed region together.
  • helper plasmid and the transposon plasmid co-transfect the target cell, the transposase produced by the helper plasmid will recognize the two ITR elements of the transposon, and then insert the transposable region and the two ITR elements into the host genome .
  • Transposon insertions usually occur at host chromosomal loci that contain TTAA sequences, with TTAA repeats flanking the transposon.
  • PiggyBac is a class II transposon that moves by a "cut-and-paste" mechanism, transposing from one place to another without leaving behind the sequence itself. Since the helper plasmid is transiently transfected into the host cell, it is gradually lost. With the loss of the helper plasmid, the transposon becomes permanently integrated in the host genome. When these host cells are transfected again with the helper plasmid, the integrated transposons are again moved by the "cut-and-paste" mechanism.
  • the selection strategy for the pool of cells using the puromycin resistance selection marker is as follows (see Figure 3):
  • Puro strategy 1 Use 10 ⁇ g/ml puromycin for screening throughout the process;
  • Puro strategy 2 Screening with increased concentration of puromycin (10 ⁇ g/ml, 50 ⁇ g/ml, 250 ⁇ g/ml);
  • GS screening markers including rat GS and human GS.
  • GS strategy 1 Use 25 ⁇ M MSX for screening throughout the process
  • GS strategy 2 use MSX with varying concentrations (25 ⁇ M, 50 ⁇ M) for screening;
  • GS strategy 3 use MSX with varying concentrations (25 ⁇ M, 50 ⁇ M, 100 ⁇ M) for screening;
  • the screening strategy for cell pools using DHFR screening markers is as follows (see Figure 5):
  • DHFR strategy 1 use 250nM methotrexate (methopterin, MTX) for screening throughout;
  • DHFR strategy 2 Screening using varying concentrations of MTX (250 nM, 500 nM).
  • Example 1 Construction of expression vectors for expressing anti-CD20 mAb (Rituximab) and screening of cell lines
  • FIG. 6 the expression vector constructed for expressing anti-CD20 mAb (Rituximab) is shown.
  • the coding sequence of the target gene expression regulatory element is shown in SEQ ID NO.1
  • the PiggyBac transposon LTD sequence is shown in SEQ ID NO.7
  • the PiggyBac transposon RTD sequence is shown in SEQ ID NO.8, bGH
  • the polyA sequence is shown in SEQ ID NO.9
  • the HSV-tk promoter sequence is shown in SEQ ID NO.10
  • the SV40 polyA sequence is shown in SEQ ID NO.11.
  • PEI 25KD 37.5 ⁇ g
  • recombinant anti-CD20 monoclonal antibody plasmid 11.25 ⁇ g see the expression vector constructed in Figure 6 for expressing anti-CD20 mAb (Rituximab)
  • transposase plasmid 1.25 ⁇ g
  • HyCell Transfx containing CHO cells -C medium GE, SH30934.04
  • the density of CHO cells in the medium is 15 ⁇ 10 6 cell/ml.
  • BalanCD CHO GROWTH A medium containing screening reagents for pressurized screening, passaging once every three days, the passaging cell density is 0.3-0.5 ⁇ 10 6 cell/ml, remove after the viability rate recovers to more than 90% Screening pressure.
  • CHO-K1 purchased from ATCC
  • CHOExpress TM purchased from ExcellGene
  • four selection markers of puromycin, rat GS, human GS, and mouse DHFR were used to construct eight expression Anti-CD20 monoclonal antibody cells, and for these cells, different cell pool screening strategies were used, as shown in Table 1 below.
  • VAV cell viability
  • the cell pool using the puromycin resistance marker and human GS marker can recover within two weeks after transfection, while the cell pool using the DHFR marker recovers much slower.
  • the concentration of screening reagents in the screening process has a certain impact on the recovery of the cell pool.
  • VCD Viable cell density
  • VIA cell viability test results of the CHO-K1 cell pool with better recovery of cell viability (as shown in FIG. 9 ).
  • VCD Viable cell density
  • VIA cell viability
  • Example 2 Fed-batch culture evaluation of cell pools using human GS and the puromycin resistance selection marker
  • the culture conditions are: 37° C., 140 rpm, 5% CO 2 , and 85% humidity.
  • Another group was set up and the temperature was adjusted to 33°C when the culture reached the 6th day, and the other conditions were the same as those described above.
  • the growth curve of the cell pool at 37°C is shown in Figure 11 .
  • the growth curve of the cell pool under the condition of cooling down to 33° C. on the sixth day is shown in FIG. 12 .
  • Example 3 Monocloning of positive cloned cells and detection of expression levels of monoclonal cell lines
  • the cells were inoculated at a density of 0.3*10 ⁇ 6cells/ml in a 250ml shake flask with a culture volume of 50ml, 140rpm, 5% CO 2 , and cultured at 37°C.
  • the cell growth curve and expression level are shown in Figure 14 and Figure 15, respectively.

Abstract

An expression vector is provided. The expression vector can be used for quick screening to obtain a cell pool, and further screening to obtain a CHO clone to generate a stable CHO cell line. The expression quantity of a target protein is improved by optimizing the regulation and control element in the expression vector.

Description

表达载体及其应用Expression vectors and their applications 技术领域technical field
本发明属于生物技术领域，具体涉及表达载体以及利用其筛选细胞池的方法。The invention belongs to the field of biotechnology, and in particular relates to an expression vector and a method for screening cell pools using it.
背景技术Background technique
中国仓鼠卵巢(CHO)细胞是制造复杂生物分子的最常用表达宿主。在CHO细胞中表达蛋白质最常用的两种方法是瞬时基因表达和稳定基因表达。瞬时基因表达是指外源基因进入受体细胞后，存在于游离的载体上，不整合到细胞的染色体上。瞬时基因表达是快速产生少量重组蛋白的首选方法，通常只需要1-3周。但是，由于需要大量的DNA和宿主细胞，在需要生成克级蛋白质的情况下，瞬时基因表达并不适用。因此，稳定基因表达通常被用来产生克级重组蛋白。稳定基因表达是指转染的目的基因整合到染色体DNA中从而表达目的蛋白。然而，稳定基因表达既耗时又费力。通常需要3-9个月才能产生CHO细胞系。因此，亟需快速、稳定、高产目的蛋白的细胞系。Chinese hamster ovary (CHO) cells are the most commonly used expression hosts for the manufacture of complex biomolecules. The two most common methods for expressing proteins in CHO cells are transient gene expression and stable gene expression. Transient gene expression means that after the exogenous gene enters the recipient cell, it exists on the free carrier and does not integrate into the chromosome of the cell. Transient gene expression is the method of choice for rapid production of small quantities of recombinant protein, usually in as little as 1-3 weeks. However, transient gene expression is not practical when gram-scale protein production is required due to the large amounts of DNA and host cells required. Therefore, stable gene expression is often used to produce gram-scale recombinant proteins. Stable gene expression refers to the integration of the transfected target gene into the chromosomal DNA to express the target protein. However, stabilizing gene expression is time-consuming and laborious. It usually takes 3-9 months to generate CHO cell lines. Therefore, there is an urgent need for cell lines that are fast, stable, and high in producing target proteins.
发明内容Contents of the invention
本发明提供了一种表达载体，通过该表达载体可以快速筛选得到细胞池，并且可通过进一步筛选得到CHO克隆，从而产生稳定的CHO细胞系。本发明中还通过对表达载体中调控元件的优化，提高了目的蛋白的表达量，快速产生的CHO细胞系不仅稳定还高产。The invention provides an expression vector, through which a cell pool can be rapidly screened, and a CHO clone can be obtained through further screening, so as to generate a stable CHO cell line. In the present invention, by optimizing the regulatory elements in the expression vector, the expression level of the target protein is increased, and the rapidly produced CHO cell line is not only stable but also highly productive.
第一方面，本发明提供了一种表达载体，所述表达载体包括筛选标记表达单元，至少一个目的基因表达单元，以及PiggyBac转座子的反向末端重复序列(ITRs)。In a first aspect, the present invention provides an expression vector, which includes a selection marker expression unit, at least one target gene expression unit, and inverted terminal repeats (ITRs) of the PiggyBac transposon.
所述筛选标记表达单元包含筛选标记的编码序列，以及与所述筛选标记的编码序列可操作连接的第一调控序列，例如，启动子的编码序列和终止子的编码序列。The screening marker expression unit comprises a coding sequence of a screening marker, and a first regulatory sequence operably linked to the coding sequence of the screening marker, for example, a coding sequence of a promoter and a coding sequence of a terminator.
在一些实施方案中，所述筛选标记包括但不限于嘌呤霉素(Puromycin,Puro)、新霉素(Neomycin,Neo)，潮霉素B(Hygromycin B,Hygro B)和杀稻瘟菌素(Blasticidin,Bsd)、谷氨酰胺合成酶(Glutamine synthetase,GS)和二氢叶酸还原酶(Dihydrofolate reductase,DHFR)。优选地，所述筛选标记为人GS。In some embodiments, the selectable markers include, but are not limited to, Puromycin (Puro), Neomycin (Neo), Hygromycin B (Hygro B), and Blasticidin ( Blasticidin, Bsd), glutamine synthetase (Glutamine synthetase, GS) and dihydrofolate reductase (Dihydrofolate reductase, DHFR). Preferably, the screening marker is human GS.
在一些实施方案中，所述筛选标记表达单元的启动子可以为单纯疱疹病毒胸腺嘧啶激酶 (Thymidine kinasegene of Herpes simplex virus,HSV-tk)启动子或猴空泡病毒40(Simian virus40,SV40)启动子。In some embodiments, the promoter of the expression unit of the selection marker can be a herpes simplex virus thymidine kinase (Thymidine kinasegene of Herpes simplex virus, HSV-tk) promoter or a simian vacuolar virus 40 (Simian virus40, SV40) promoter son.
在一些实施方案中，所述筛选标记表达单元的终止子可以为聚腺苷化信号(PolyA)，例如SV40 PolyA，牛生长激素(Bovine growth hormone，bGH)PolyA，HSV-tk PolyA或兔β-珠蛋白(Rabbit beta-globin，RBG)PolyA，优选为SV40 PolyA。In some embodiments, the terminator of the selection marker expression unit can be a polyadenylation signal (PolyA), such as SV40 PolyA, bovine growth hormone (Bovine growth hormone, bGH) PolyA, HSV-tk PolyA or rabbit β- Globin (Rabbit beta-globin, RBG) PolyA, preferably SV40 PolyA.
优选地，所述筛选标记表达单元包含HSV-tk启动子的编码序列和SV40 polyA的编码序列。Preferably, the screening marker expression unit comprises the coding sequence of HSV-tk promoter and the coding sequence of SV40 polyA.
优选地，所述筛选标记表达单元包含HSV-tk启动子的编码序列，人GS筛选标记的编码序列和SV40 polyA的编码序列。Preferably, the screening marker expression unit comprises the coding sequence of the HSV-tk promoter, the coding sequence of the human GS screening marker and the coding sequence of SV40 polyA.
所述目的基因表达单元包含目的基因序列，以及与目的基因序列可操作连接的第二调控序列，例如，目的基因表达调控元件的编码序列和终止子的编码序列。The target gene expression unit comprises the target gene sequence and a second regulatory sequence operably linked to the target gene sequence, for example, the coding sequence of the target gene expression regulatory element and the coding sequence of the terminator.
在一些实施方案中，所述目的基因表达调控元件的编码序列调控序列具有选自SEQ ID NO.1-6所示的序列。In some embodiments, the coding sequence regulatory sequence of the target gene expression regulatory element has a sequence selected from the sequences shown in SEQ ID NO.1-6.
在一些实施方案中，所述目的基因表达单元的终止子可以为polyA，例如SV40 PolyA，bGH PolyA，tk PolyA，RBG PolyA，优选为bGH PolyA。In some embodiments, the terminator of the target gene expression unit can be polyA, such as SV40 PolyA, bGH PolyA, tk PolyA, RBG PolyA, preferably bGH PolyA.
在一些实施方案中，所述目的基因为一个或者多个，例如2个，3个或者4个，相应地，所述表达载体具有2个，3个或者4个目的基因表达单元。In some embodiments, the target gene is one or more, such as 2, 3 or 4, and correspondingly, the expression vector has 2, 3 or 4 target gene expression units.
在一些实施方案中，所述目的基因编码anti-CD20单克隆抗体(Rituximab)或anti-HER2单克隆抗体(Trastuzumab)的轻链和/或重链。相应地，所述表达载体具有1个或者2个目的基因表达单元。In some embodiments, the target gene encodes the light chain and/or heavy chain of an anti-CD20 monoclonal antibody (Rituximab) or an anti-HER2 monoclonal antibody (Trastuzumab). Correspondingly, the expression vector has 1 or 2 target gene expression units.
在一些实施方案中，所述表达载体可以快速筛选获得CHO细胞池，进一步筛选得到CHO克隆产生稳定CHO细胞系，还通过对表达载体中调控元件的优化，提高了目的蛋白的表达量，快速产生的CHO细胞系不仅稳定还高产。In some embodiments, the expression vector can be quickly screened to obtain a CHO cell pool, and further screened to obtain a CHO clone to generate a stable CHO cell line, and by optimizing the regulatory elements in the expression vector, the expression of the target protein is increased, and the rapid production The CHO cell line is not only stable but also highly productive.
第二方面，本发明提供了含有第一方面的表达载体的表达系统。In a second aspect, the present invention provides an expression system comprising the expression vector of the first aspect.
在一些实施方案中，所述表达系统还包含辅助质粒，所述辅助质粒编码PiggyBac转座酶。In some embodiments, the expression system further comprises a helper plasmid encoding a PiggyBac transposase.
第三方面，本发明提供了宿主细胞，所述宿主细胞转染有第一方面的表达载体。In a third aspect, the present invention provides host cells transfected with the expression vector of the first aspect.
在一些实施方案中，所述宿主细胞转染有第二方面的表达系统。In some embodiments, the host cell is transfected with the expression system of the second aspect.
优选地，所述宿主细胞的基因组中稳定整合有第一方面的表达载体。Preferably, the expression vector of the first aspect is stably integrated in the genome of the host cell.
在一些实施方案中，所述宿主细胞不包含编码PiggyBac转座酶的辅助质粒。In some embodiments, the host cell does not contain a helper plasmid encoding a PiggyBac transposase.
在一些实施方案中，所述宿主细胞为CHO细胞，例如为CHO-K1细胞或CHOExpress TM细胞。 In some embodiments, the host cell is a CHO cell, such as a CHO-K1 cell or a CHOExpress ™ cell.
在一些实施方案中，所述宿主细胞为稳定的CHO细胞系。In some embodiments, the host cell is a stable CHO cell line.
在一些实施方案中，所述宿主细胞具有提高的目的蛋白表达量。In some embodiments, the host cell has increased expression of the protein of interest.
第四方面，本发明提供了利用第一方面的表达载体或第二方面的表达系统筛选细胞的方法，所述方法包括以下步骤：In a fourth aspect, the present invention provides a method for screening cells using the expression vector of the first aspect or the expression system of the second aspect, the method comprising the following steps:
(a)用第一方面的表达载体或第二方面的表达系统转染宿主细胞；以及(a) transfecting host cells with the expression vector of the first aspect or the expression system of the second aspect; and
(b)利用针对所述表达载体中的筛选标记的筛选试剂对步骤(a)获得的宿主细胞进行筛选的步骤。(b) a step of screening the host cells obtained in step (a) with a screening reagent directed against the screening marker in the expression vector.
在一些实施方案中，所述宿主细胞为CHO细胞，例如为CHO-K1细胞或CHOExpress TM细胞。 In some embodiments, the host cell is a CHO cell, such as a CHO-K1 cell or a CHOExpress ™ cell.
在一些实施方案中，所述筛选标记为嘌呤霉素，并且使用10μg/ml的嘌呤霉素对宿主细胞进行筛选，所述宿主细胞转染有包含嘌呤霉素抗性筛选标记的所述表达载体。In some embodiments, the selection marker is puromycin, and 10 μg/ml of puromycin is used to select host cells transfected with the expression vector comprising a puromycin resistance selection marker .
在一些实施方案中，所述筛选标记为嘌呤霉素，并且使用浓度增高的嘌呤霉素(10μg/ml、50μg/ml、250μg/ml)对宿主细胞进行筛选，所述宿主细胞转染有包含嘌呤霉素抗性筛选标记的所述表达载体。In some embodiments, the selection marker is puromycin, and increased concentrations of puromycin (10 μg/ml, 50 μg/ml, 250 μg/ml) are used to select host cells transfected with The expression vector for the puromycin resistance selection marker.
在一些实施方案中，所述筛选标记为GS，并且使用25μM的蛋氨酸亚氨基代砜(methionine sulfoximine,MSX)对宿主细胞进行筛选，所述宿主细胞转染有包含GS筛选标记的所述表达载体。In some embodiments, the selection marker is GS, and 25 μM methionine sulfoximine (MSX) is used to select host cells transfected with the expression vector comprising the GS selection marker .
在一些实施方案中，所述筛选标记为GS，并且使用浓度增高的MSX(25μM、50μM)对宿主细胞进行筛选，所述宿主细胞转染有包含GS筛选标记的所述表达载体。In some embodiments, the selection marker is GS, and an increased concentration of MSX (25 μM, 50 μM) is used to select host cells transfected with the expression vector comprising the GS selection marker.
在一些实施方案中，所述筛选标记为GS，并且使用浓度增高的MSX(25μM、50μM、100μM)对宿主细胞进行筛选，所述宿主细胞转染有包含GS筛选标记的所述表达载体。In some embodiments, the selection marker is GS, and increased concentrations of MSX (25 μM, 50 μM, 100 μM) are used to select host cells transfected with the expression vector comprising the GS selection marker.
优选地，所述筛选标记为人GS。Preferably, the screening marker is human GS.
在一些实施方案中，所述筛选标记为DHFR，并且使用250nM的氨甲喋呤(amethopterin,MTX)对宿主细胞进行筛选，所述宿主细胞转染有包含DHFR筛选标记的所述表达载体。In some embodiments, the selection marker is DHFR, and 250 nM of methotrexate (methopterin, MTX) is used to select host cells transfected with the expression vector comprising the DHFR selection marker.
在一些实施方案中，所述筛选标记为DHFR，并且使用浓度增高的氨甲喋呤(250nM、500nM)对宿主细胞进行筛选，所述宿主细胞转染有包含DHFR筛选标记的所述表达载体。In some embodiments, the selection marker is DHFR, and increased concentrations of methotrexate (250 nM, 500 nM) are used to select host cells transfected with the expression vector comprising the DHFR selection marker.
优选地，所述筛选试剂为嘌呤霉素，蛋氨酸亚氨基代砜或氨甲喋呤。Preferably, the screening reagent is puromycin, methionine iminosulfone or methotrexate.
在一些实施方案中，用含有嘌呤霉素，蛋氨酸亚氨基代砜或氨甲喋呤的CHO细胞培养基对所述宿主细胞进行加压筛选。在一些实施方案中，所述培养基为BalanCD CHO GROWTH A培养基。In some embodiments, the host cells are subjected to pressure selection with CHO cell culture medium containing puromycin, methionine sulfone imino, or methotrexate. In some embodiments, the medium is BalanCD CHO GROWTH A medium.
在一些实施方案中，待活率恢复至90％以上后撤去筛选压力。In some embodiments, the screening pressure is removed after the viability rate recovers to above 90%.
本发明的筛选方法筛选得到高产稳定的CHO克隆，进而产生稳定的CHO细胞系。The screening method of the present invention screens high-yield and stable CHO clones, and then produces stable CHO cell lines.
第五方面，本发明提供了由第四方面的筛选方法获得的细胞。In a fifth aspect, the present invention provides cells obtained by the screening method of the fourth aspect.
第六方面，本发明提供了第一方面的表达载体，第二方面的表达系统，第三方面的宿主细胞，或第四方面的筛选方法，或第五方面的细胞在生产表达量提高的目的蛋白中的应用。In the sixth aspect, the present invention provides the expression vector of the first aspect, the expression system of the second aspect, the host cell of the third aspect, or the screening method of the fourth aspect, or the purpose of increasing the expression level of the cells of the fifth aspect protein application.
本发明中提供的表达载体，通过该表达载体可以快速筛选得到细胞池，并且可通过进一步筛选得到CHO克隆产生稳定CHO细胞系，大大缩短了产生稳定CHO细胞系的时间，同时还通过对表达载体中调控元件的优化，提高了目的蛋白的表达量，快速产生的CHO细胞系不仅稳定还高产。The expression vector provided in the present invention can be quickly screened to obtain a cell pool through the expression vector, and can be further screened to obtain a CHO clone to produce a stable CHO cell line, which greatly shortens the time for producing a stable CHO cell line. The optimization of the regulatory elements in the medium increases the expression of the target protein, and the rapidly produced CHO cell line is not only stable but also highly productive.
附图说明：Description of drawings:
图1显示了包含两个目的基因表达单元的表达载体结构图。Figure 1 shows a structural diagram of an expression vector comprising two target gene expression units.
图2显示了包含一个目的基因表达单元的表达载体结构图。Figure 2 shows the structural diagram of an expression vector comprising an expression unit of a target gene.
图3显示了针对使用嘌呤霉素抗性筛选标记的细胞池的筛选策略。Figure 3 shows the selection strategy against the pool of cells using the puromycin resistance selection marker.
图4显示了针对使用GS筛选标记(包括大鼠GS以及人GS)的细胞池的筛选策略。Figure 4 shows the screening strategy for cell pools using GS selection markers including rat GS as well as human GS.
图5显示了针对使用DHFR筛选标记的细胞池的筛选策略。Figure 5 shows the screening strategy against the cell pool using the DHFR selection marker.
图6显示了用于表达anti-CD20 mAb(Rituximab)的表达载体结构。Figure 6 shows the expression vector structure for expressing anti-CD20 mAb (Rituximab).
图7显示了CHO-K1细胞池的细胞活率(VIA)。Figure 7 shows the cell viability (VIA) of the CHO-K1 cell pool.
图8显示了CHOExpress TM细胞池的细胞活率(VIA)。 Figure 8 shows the cell viability (VIA) of the CHOExpress ™ cell pool.
图9显示了细胞活率恢复较好的CHO-K1细胞池的活细胞密度(VCD)和细胞活率(VIA)。Figure 9 shows the viable cell density (VCD) and cell viability (VIA) of the CHO-K1 cell pool with better recovery of cell viability.
图10显示了细胞活率恢复较好的CHOExpress TM细胞池的活细胞密度(VCD)和细胞活率(VIA)。 Figure 10 shows the viable cell density (VCD) and cell viability (VIA) of the CHOExpress ™ cell pool with better recovery of cell viability.
图11显示了37℃条件下细胞池的生长曲线，其中，“KP10”指K1-PR10，“KP250”指K1-PR250，“EP10”指EXP-PR10，“EP250”指EXP-PR250，“KH25”指K1-HG25，“KH50”指K1-HG50，“KH100”指K1-HG100，“EH25”指EXP-HG25，“EH50”指EXP-HG50，“EH100”指EXP-HG100。Figure 11 shows the growth curve of the cell pool at 37°C, where "KP10" refers to K1-PR10, "KP250" refers to K1-PR250, "EP10" refers to EXP-PR10, "EP250" refers to EXP-PR250, "KH25 " refers to K1-HG25, "KH50" refers to K1-HG50, "KH100" refers to K1-HG100, "EH25" refers to EXP-HG25, "EH50" refers to EXP-HG50, and "EH100" refers to EXP-HG100.
图12显示了第六天降温至33℃条件下细胞池的生长曲线，其中，“KP10”指K1-PR10，“KP250”指K1-PR250，“EP10”指EXP-PR10，“EP250”指EXP-PR250，“KH25”指K1-HG25，“KH50”指K1-HG50，“KH100”指K1-HG100，“EH25”指EXP-HG25，“EH50”指EXP-HG50，“EH100”指EXP-HG100。Figure 12 shows the growth curve of the cell pool when the temperature was lowered to 33°C on the sixth day, where "KP10" refers to K1-PR10, "KP250" refers to K1-PR250, "EP10" refers to EXP-PR10, and "EP250" refers to EXP -PR250, "KH25" refers to K1-HG25, "KH50" refers to K1-HG50, "KH100" refers to K1-HG100, "EH25" refers to EXP-HG25, "EH50" refers to EXP-HG50, "EH100" refers to EXP-HG100 .
图13显示了Protein A HPLC检测表达量结果，其中，“KP10”指K1-PR10，“KP250”指K1-PR250，“EP10”指EXP-PR10，“EP250”指EXP-PR250，“KH25”指K1-HG25，“KH50”指K1-HG50，“KH100”指K1-HG100，“EH25”指EXP-HG25，“EH50”指EXP-HG50，“EH100”指EXP-HG100。Figure 13 shows the results of Protein A HPLC detection expression level, where "KP10" refers to K1-PR10, "KP250" refers to K1-PR250, "EP10" refers to EXP-PR10, "EP250" refers to EXP-PR250, "KH25" refers to K1-HG25, "KH50" refers to K1-HG50, "KH100" refers to K1-HG100, "EH25" refers to EXP-HG25, "EH50" refers to EXP-HG50, and "EH100" refers to EXP-HG100.
图14显示了实施例3中细胞的生长曲线。Figure 14 shows the growth curve of the cells in Example 3.
图15显示了实施例3中表达量检测结果。FIG. 15 shows the expression detection results in Example 3.
图16显示了实施例4中的表达量检测结果。FIG. 16 shows the expression detection results in Example 4.
具体实施方式Detailed ways
在一些实施方案中，本发明可以构建包含两个目的基因表达单元的表达载体(参见图1)，或者构建包含一个目的基因表达单元的表达载体(参见图2)。In some embodiments, the present invention can construct an expression vector comprising two target gene expression units (see FIG. 1 ), or construct an expression vector comprising one target gene expression unit (see FIG. 2 ).
本发明采用的PiggyBac系统包含两个载体，一个载体被称为辅助质粒，负责编码转座酶；另一个载体被称为转座子质粒，包含两个末端重复序列(ITRs)以及两者之间的被转座区域，启动子、表达盒和目的基因一起被克隆到这个转座区域。当辅助质粒和转座子质粒共转染靶细胞时，辅助质粒产生的转座酶将会识别转座子的两个ITR元件，然后将被转座区和两个ITR元件插入到宿主基因组中。转座插入通常发生在包含TTAA序列的宿主染色体位点，并在转座子两侧出现TTAA重复序列。The PiggyBac system that the present invention adopts comprises two vectors, and a vector is called helper plasmid, is responsible for coding transposase; Another vector is called transposon plasmid, comprises two terminal repeats (ITRs) and between the two The transposed region, promoter, expression cassette and gene of interest are cloned into this transposed region together. When the helper plasmid and the transposon plasmid co-transfect the target cell, the transposase produced by the helper plasmid will recognize the two ITR elements of the transposon, and then insert the transposable region and the two ITR elements into the host genome . Transposon insertions usually occur at host chromosomal loci that contain TTAA sequences, with TTAA repeats flanking the transposon.
PiggyBac属于II类转座子，通过“剪切—粘贴”的机制移动，从一个地方转座到另一个地方，而不留下序列本身。由于辅助质粒是通过瞬时转染进入宿主细胞的，故会逐渐丢失。随着辅助质粒的丢失，转座子在宿主基因组中变成了永久整合。当这些宿主细胞再次被辅助质粒转 染，整合的转座子会再次通过“剪切—粘贴”的机制移动。PiggyBac is a class II transposon that moves by a "cut-and-paste" mechanism, transposing from one place to another without leaving behind the sequence itself. Since the helper plasmid is transiently transfected into the host cell, it is gradually lost. With the loss of the helper plasmid, the transposon becomes permanently integrated in the host genome. When these host cells are transfected again with the helper plasmid, the integrated transposons are again moved by the "cut-and-paste" mechanism.
在利用本发明的表达载体或本发明的表达系统筛选细胞池的方法中，In the method for screening cell pools using the expression vector of the present invention or the expression system of the present invention,
使用嘌呤霉素抗性筛选标记的细胞池的筛选策略如下(参见图3)：The selection strategy for the pool of cells using the puromycin resistance selection marker is as follows (see Figure 3):
puro策略1：全程使用10μg/ml的嘌呤霉素进行筛选；Puro strategy 1: Use 10 μg/ml puromycin for screening throughout the process;
puro策略2：使用浓度增高的嘌呤霉素(10μg/ml、50μg/ml、250μg/ml)进行筛选；Puro strategy 2: Screening with increased concentration of puromycin (10 μg/ml, 50 μg/ml, 250 μg/ml);
使用GS筛选标记(包括大鼠GS以及人GS)的细胞池的筛选策略如下(参见图4)：The screening strategy for cell pools using GS screening markers (including rat GS and human GS) is as follows (see Figure 4):
GS策略1：全程使用25μM的MSX进行筛选；GS strategy 1: Use 25 μM MSX for screening throughout the process;
GS策略2：使用浓度变化的MSX(25μM、50μM)进行筛选；GS strategy 2: use MSX with varying concentrations (25 μM, 50 μM) for screening;
GS策略3：使用浓度变化的MSX(25μM、50μM、100μM)进行筛选；GS strategy 3: use MSX with varying concentrations (25 μM, 50 μM, 100 μM) for screening;
使用DHFR筛选标记的细胞池的筛选策略如下(参见图5)：The screening strategy for cell pools using DHFR screening markers is as follows (see Figure 5):
DHFR策略1：全程使用250nM的氨甲喋呤(amethopterin,MTX)进行筛选；DHFR strategy 1: use 250nM methotrexate (methopterin, MTX) for screening throughout;
DHFR策略2：使用浓度变化的MTX(250nM、500nM)进行筛选。DHFR strategy 2: Screening using varying concentrations of MTX (250 nM, 500 nM).
为使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明，并不用于构成对本发明的任何限制。In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. The specific embodiments described here are only used to explain the present invention, and are not intended to constitute any limitation to the present invention.
实施例1：用于表达anti-CD20 mAb(Rituximab)的表达载体构建以及细胞株的筛选Example 1: Construction of expression vectors for expressing anti-CD20 mAb (Rituximab) and screening of cell lines
CHO细胞系的构建与筛选Construction and screening of CHO cell lines
参见图6，显示了构建用于表达anti-CD20 mAb(Rituximab)的表达载体。Referring to Fig. 6, the expression vector constructed for expressing anti-CD20 mAb (Rituximab) is shown.
其中目的基因表达调控元件的编码序列如SEQ ID NO.1所示，PiggyBac转座子LTD序列如SEQ ID NO.7所示，其中PiggyBac转座子RTD序列如SEQ ID NO.8所示，bGH polyA序列如SEQ ID NO.9所示，HSV-tk启动子序列如SEQ ID NO.10所示，SV40 polyA序列如SEQ ID NO.11所示。The coding sequence of the target gene expression regulatory element is shown in SEQ ID NO.1, the PiggyBac transposon LTD sequence is shown in SEQ ID NO.7, and the PiggyBac transposon RTD sequence is shown in SEQ ID NO.8, bGH The polyA sequence is shown in SEQ ID NO.9, the HSV-tk promoter sequence is shown in SEQ ID NO.10, and the SV40 polyA sequence is shown in SEQ ID NO.11.
转染体系：Transfection system:
PEI(25KD)37.5μg；重组抗CD20单克隆抗体质粒11.25μg(参见图6构建的用于表达anti-CD20 mAb(Rituximab)的表达载体)；转座酶质粒1.25μg；含有CHO细胞的HyCell Transfx-C培养基(GE,SH30934.04)1.5ml；CHO细胞在培养基中的密度15×10 6cell/ml。 PEI (25KD) 37.5 μg; recombinant anti-CD20 monoclonal antibody plasmid 11.25 μg (see the expression vector constructed in Figure 6 for expressing anti-CD20 mAb (Rituximab)); transposase plasmid 1.25 μg; HyCell Transfx containing CHO cells -C medium (GE, SH30934.04) 1.5ml; the density of CHO cells in the medium is 15×10 6 cell/ml.
转染方法：Transfection method:
转染时，将PEI、重组抗CD20单克隆抗体质粒、转座酶质粒及含有CHO细胞的HyCell Transfx-C培养基混匀后放入37℃摇床孵育，转染1小时后加入3.5ml BalanCD CHO GROWTH A。For transfection, mix PEI, recombinant anti-CD20 monoclonal antibody plasmids, transposase plasmids and HyCell Transfx-C medium containing CHO cells and incubate on a shaker at 37°C, add 3.5ml BalanCD 1 hour after transfection CHO GROWTH A.
筛选方法：Screening method:
转染两天后，用含有筛选试剂的BalanCD CHO GROWTH A medium进行加压筛选，每三天传代一次，传代细胞密度为0.3-0.5×10 6cell/ml，待活率恢复至90％以上后撤去筛选压力。 Two days after transfection, use BalanCD CHO GROWTH A medium containing screening reagents for pressurized screening, passaging once every three days, the passaging cell density is 0.3-0.5×10 6 cell/ml, remove after the viability rate recovers to more than 90% Screening pressure.
本实施例中使用CHO-K1(购于ATCC)和CHOExpress TM(购于ExcellGene)两种宿主细胞，以及嘌呤霉素、大鼠GS、人GS、小鼠DHFR四种筛选标记构建了八种表达抗CD20单克隆抗体的细胞，并针对这些细胞，采用了不同的细胞池筛选策略，具体如下表1所示。 In this example, two host cells, CHO-K1 (purchased from ATCC) and CHOExpress ™ (purchased from ExcellGene), and four selection markers of puromycin, rat GS, human GS, and mouse DHFR were used to construct eight expression Anti-CD20 monoclonal antibody cells, and for these cells, different cell pool screening strategies were used, as shown in Table 1 below.
表1：Table 1:
组别group 筛选标记filter marker 细胞系cell line 筛选策略screening strategy
K1-PR10K1-PR10 嘌呤霉素抗性筛选标记Puromycin resistance selection marker CHO-K1CHO-K1 Puro策略1Puro strategy 1
K1-PR250K1-PR250 嘌呤霉素抗性筛选标记Puromycin resistance selection marker CHO-K1CHO-K1 Puro策略2Puro strategy 2
K1-RG25K1-RG25 大鼠GSRat GS CHO-K1CHO-K1 GS策略1GS Strategy 1
K1-RG50K1-RG50 大鼠GSRat GS CHO-K1CHO-K1 GS策略2GS Strategy 2
K1-RG100K1-RG100 大鼠GSRat GS CHO-K1CHO-K1 GS策略3GS strategy 3
K1-HG25K1-HG25 人GSHuman GS CHO-K1CHO-K1 GS策略1GS Strategy 1
K1-HG50K1-HG50 人GSHuman GS CHO-K1CHO-K1 GS策略2GS Strategy 2
K1-HG100K1-HG100 人GSHuman GS CHO-K1CHO-K1 GS策略3GS strategy 3
K1-MD250K1-MD250 小鼠DHFRmouseDHFR CHO-K1CHO-K1 DHFR策略1DHFR strategy 1
K1-MD500K1-MD500 小鼠DHFRmouseDHFR CHO-K1CHO-K1 DHFR策略2DHFR Strategy 2
EXP-PR10EXP-PR10 嘌呤霉素抗性筛选标记Puromycin resistance selection marker CHOExpress TM CHOExpress ™ Puro策略1Puro strategy 1
EXP-PR250EXP-PR250 嘌呤霉素抗性筛选标记Puromycin resistance selection marker CHOExpress TM CHOExpress ™ Puro策略2Puro strategy 2
EXP-RG25EXP-RG25 大鼠GSRat GS CHOExpress TM CHOExpress ™ GS策略1GS Strategy 1
EXP-RG50EXP-RG50 大鼠GSRat GS CHOExpress TM CHOExpress ™ GS策略2GS Strategy 2
EXP-RG100EXP-RG100 大鼠GSRat GS CHOExpress TM CHOExpress ™ GS策略3GS strategy 3
EXP-HG25EXP-HG25 人GSHuman GS CHOExpress TM CHOExpress ™ GS策略1GS Strategy 1
EXP-HG50EXP-HG50 人GSHuman GS CHOExpress TM CHOExpress ™ GS策略2GS Strategy 2
EXP-HG100EXP-HG100 人GSHuman GS CHOExpress TM CHOExpress ™ GS策略3GS strategy 3
EXP-MD250EXP-MD250 小鼠DHFRmouseDHFR CHOExpress TM CHOExpress ™ DHFR策略1DHFR strategy 1
EXP-MD500EXP-MD500 小鼠DHFRmouseDHFR CHOExpress TM CHOExpress ™ DHFR策略2DHFR Strategy 2
CHO-K1细胞池的细胞活率(viability,VIA)如图7和下表2所示。The cell viability (VIA) of the CHO-K1 cell pool is shown in Figure 7 and Table 2 below.
表2：Table 2:
 the Day 11 Day 11 Day 14 Day 14 Day 17 Day 17
K1-HG25K1-HG25 90.790.7 94.7894.78 93.1693.16
K1-HG50K1-HG50 87.8787.87 94.0894.08 93.993.9
K1-HG100K1-HG100 87.8787.87 89.0289.02 88.9688.96
K1-MD250K1-MD250 40.2640.26 49.2949.29 66.7566.75
K1-MD500K1-MD500 30.1930.19 41.6941.69 48.6448.64
K1-PR10K1-PR10 94.7194.71 97.0497.04 96.5896.58
K1-PR250K1-PR250 60.0660.06 88.2588.25 88.6888.68
K1-RG25K1-RG25 68.0768.07 77.3777.37 84.9384.93
K1-RG50K1-RG50 63.8563.85 76.9976.99 81.0881.08
K1-RG100K1-RG100 63.8563.85 72.5372.53 75.9675.96
由上述结果可知，采用嘌呤霉素抗性标记和人GS标记的细胞池可以在在转染后两周内恢复，使用DHFR标记的细胞池则恢复的慢很多。另外，筛选过程中的筛选试剂的浓度对细胞池的恢复有一定影响。From the above results, it can be seen that the cell pool using the puromycin resistance marker and human GS marker can recover within two weeks after transfection, while the cell pool using the DHFR marker recovers much slower. In addition, the concentration of screening reagents in the screening process has a certain impact on the recovery of the cell pool.
CHOExpress TM细胞池的细胞活率如图8和下表3所示： The cell viability of the CHOExpress TM cell pool is shown in Figure 8 and Table 3 below:
表3：table 3:
 the Day 11 Day 11 Day 14 Day 14 Day 17 Day 17
EXP-HG25EXP-HG25 95.495.4 97.6797.67 97.8697.86
EXP-HG50EXP-HG50 97.1297.12 95.8495.84 97.2897.28
EXP-HG100EXP-HG100 97.1297.12 91.8291.82 92.1192.11
EXP-MD250EXP-MD250 24.5824.58 36.9936.99 35.8235.82
EXP-MD500EXP-MD500 24.1424.14 24.6324.63 62.6262.62
EXP-PR10EXP-PR10 98.5998.59 99.2899.28 99.2499.24
EXP-PR250EXP-PR250 77.8677.86 95.8295.82 89.5489.54
EXP-RG25EXP-RG25 68.9168.91 79.4379.43 84.1184.11
EXP-RG50EXP-RG50 54.7654.76 60.6860.68 75.3875.38
EXP-RG100EXP-RG100 54.7654.76 59.8959.89 63.1963.19
细胞活率恢复较好的CHO-K1细胞池的活细胞密度(VCD)及细胞活率(VIA)检测结果(如图9所示)。Viable cell density (VCD) and cell viability (VIA) test results of the CHO-K1 cell pool with better recovery of cell viability (as shown in FIG. 9 ).
细胞活率恢复较好的CHOExpress TM细胞池的活细胞密度(VCD)及细胞活率(VIA)检测结果(如图10所示)。 Viable cell density (VCD) and cell viability (VIA) detection results of the CHOExpress ™ cell pool with better recovery of cell viability (as shown in FIG. 10 ).
实施例2：补料批培养评估使用人GS以及嘌呤霉素抗性筛选标记的细胞池Example 2: Fed-batch culture evaluation of cell pools using human GS and the puromycin resistance selection marker
将筛选结束的细胞以0.5×10 6cell/ml(seed by centrifuge)的细胞密度接种到50ml含有0.2％抗结团剂(ACA)EX-CELL Advanced CHO Fed-batch培养基中，混匀后放入摇床培养。培养条件为：37℃,140rpm,5％CO 2,85％湿度。 Inoculate the screened cells into 50ml EX-CELL Advanced CHO Fed-batch medium containing 0.2% anti-caking agent (ACA) at a cell density of 0.5×10 6 cell/ml (seed by centrifuge), mix well and put cultured in a shaker. The culture conditions are: 37° C., 140 rpm, 5% CO 2 , and 85% humidity.
补料策略：Feed strategy:
在培养的第2、4、6、8、10天加入3％BalanCD CHO Feed 4+0.3％Cell Boost 7b；Add 3% BalanCD CHO Feed 4+0.3% Cell Boost 7b on the 2nd, 4th, 6th, 8th, and 10th day of culture;
当葡萄糖浓度低于4g/L时，添加葡萄糖至其浓度为8g/L；When the glucose concentration is lower than 4g/L, add glucose to its concentration of 8g/L;
对于使用嘌呤霉素抗性筛选标记的细胞池，在培养的第2-5天，保持谷氨酰胺浓度不低于4mM。For cell pools using the puromycin resistance selection marker, keep the glutamine concentration not lower than 4mM on days 2-5 of culture.
另外设置一组在培养至第6天时将温度调至33℃，其他条件与上述描述相同。Another group was set up and the temperature was adjusted to 33°C when the culture reached the 6th day, and the other conditions were the same as those described above.
37℃条件下细胞池的生长曲线如图11所示。The growth curve of the cell pool at 37°C is shown in Figure 11 .
第六天降温至33℃条件下细胞池的生长曲线如图12所示。The growth curve of the cell pool under the condition of cooling down to 33° C. on the sixth day is shown in FIG. 12 .
通过上述图12的生长曲线可知，第六天降温至33℃与一直保持在37℃细胞生长趋势接近，但是降温至33℃的情况下，细胞可以存活更长时间。From the above growth curve in Figure 12, it can be seen that cooling to 33°C on the sixth day is close to the growth trend of cells kept at 37°C, but the cells can survive for a longer time when the temperature is lowered to 33°C.
Protein A HPLC检测表达量结果如图13所示。The results of protein A HPLC detection expression level are shown in Figure 13.
实施例3：阳性克隆细胞的单克隆化与单克隆细胞株表达量检测Example 3: Monocloning of positive cloned cells and detection of expression levels of monoclonal cell lines
通过有限稀释法从表达量最高的细胞池中挑取单克隆，最后针对每个单克隆扩大培养，用Octet分子互作仪检测培养上清中的目标蛋白表达量，得到高产的单克隆细胞株。Single clones were selected from the cell pool with the highest expression by the limiting dilution method, and finally each single clone was expanded for culture, and the expression of the target protein in the culture supernatant was detected with an Octet molecular interaction instrument to obtain a high-yielding monoclonal cell line .
取上述步骤中筛选出的阳性单克隆细胞株，以Advanced CHO Fed-batch为基础培养基，Cell Boost 7a/7b为补料培养基进行补料批培养阳性单克隆细胞株。细胞表达量评估时，按0.3*10^6cells/ml的密度接种于250ml摇瓶中进行，培养体积为50ml，140rpm，5％CO 2，37℃培养。 Take the positive monoclonal cell lines screened in the above steps, and use Advanced CHO Fed-batch as the basal medium and Cell Boost 7a/7b as the feeding medium to carry out fed-batch culture of the positive monoclonal cell lines. When evaluating the expression level of cells, the cells were inoculated at a density of 0.3*10^6cells/ml in a 250ml shake flask with a culture volume of 50ml, 140rpm, 5% CO 2 , and cultured at 37°C.
补料策略：Feed strategy:
细胞接种第3天起每天补充3％Cell Boost 7a+0.3％Cell Boost 7b。 Supplement 3% Cell Boost 7a+0.3% Cell Boost 7b every day from the 3rd day of cell inoculation.
当葡萄糖浓度低于5g/L时，添加葡萄糖至其浓度为8g/L。When the glucose concentration is lower than 5g/L, add glucose to a concentration of 8g/L.
细胞生长曲线和表达量分别如图14和图15所示。The cell growth curve and expression level are shown in Figure 14 and Figure 15, respectively.
实施例4Example 4
采用本发明实施例中的表达载体构建anti-HER2单克隆抗体(Trastuzumab)表达载体(人GS筛选标记)，经过转染(CHO-K1为宿主细胞)、细胞池筛选(GS策略3)及阳性克隆细胞的单克隆化得到单克隆细胞株。取筛选出的阳性单克隆细胞株，以HyClone Actipro细胞培养基为基础培养基，Cell Boost 7a/7b为补料培养基进行补料批培养阳性单克隆细胞株。细胞表 达量评估时，按10*10^6cells/ml的密度接种于250ml摇瓶中进行，培养体积为50ml，140rpm，5％CO 2，37℃培养。 Use the expression vector in the embodiment of the present invention to construct anti-HER2 monoclonal antibody (Trastuzumab) expression vector (human GS screening marker), after transfection (CHO-K1 is the host cell), cell pool screening (GS strategy 3) and positive Monoclonalization of cloned cells yields monoclonal cell lines. Take the screened positive monoclonal cell lines, use HyClone Actipro cell culture medium as the basal medium, and Cell Boost 7a/7b as the feed medium to carry out fed-batch culture of the positive monoclonal cell lines. When evaluating the expression level of cells, inoculate in a 250ml shake flask at a density of 10*10^6cells/ml, culture in a volume of 50ml, 140rpm, 5% CO 2 , and culture at 37°C.
补料策略：Feed strategy:
初始添加2％的Cell Boost 7a和2％的Cell Boost 7b；Initially add 2% Cell Boost 7a and 2% Cell Boost 7b;
细胞VCD＞20*10^6cells/ml时，添加4％的Cell Boost 7a和4％的Cell Boost 7b；When cell VCD>20*10^6cells/ml, add 4% Cell Boost 7a and 4% Cell Boost 7b;
细胞VCD＞30*10^6cells/ml时，添加3％的Cell Boost 7a和0.6％的Cell Boost 7b；When cell VCD>30*10^6cells/ml, add 3% Cell Boost 7a and 0.6% Cell Boost 7b;
当葡萄糖浓度低于5g/L时，添加葡萄糖至其浓度为8g/L。When the glucose concentration is lower than 5g/L, add glucose to a concentration of 8g/L.
表达量如图16所示。The expression levels are shown in Figure 16.
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容本发明的技术方案不限于上述具体实施例的限制，凡是根据本发明的技术方案做出的技术变形，均落入本发明的保护范围之内。In addition, various combinations of different implementations of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, it should also be regarded as the disclosed content of the present invention. The technical solution of the present invention is not limited to the above-mentioned specific examples All technical modifications made according to the technical solutions of the present invention fall within the scope of protection of the present invention.

Claims (12)

  1. 一种表达载体，所述表达载体包括筛选标记表达单元，至少一个目的基因表达单元，以及PiggyBac转座子的反向末端重复序列。An expression vector, comprising a selection marker expression unit, at least one target gene expression unit, and an inverted terminal repeat sequence of a PiggyBac transposon.
  2. 根据权利要求1的表达载体，其中，所述筛选标记表达单元包含筛选标记的编码序列，以及与所述筛选标记的编码序列可操作连接的第一调控序列，优选地，所述第一调控序列包括启动子的编码序列和终止子的编码序列。The expression vector according to claim 1, wherein said screening marker expression unit comprises a coding sequence of a screening marker, and a first regulatory sequence operably linked to the coding sequence of said screening marker, preferably said first regulatory sequence Include the coding sequence of the promoter and the coding sequence of the terminator.
  3. 根据权利要求2的表达载体，其中，所述筛选标记为嘌呤霉素、新霉素、潮霉素B、和杀稻瘟菌素、谷氨酰胺合成酶、或二氢叶酸还原酶，优选地，所述筛选标记为人谷氨酰胺合成酶。The expression vector according to claim 2, wherein the selection marker is puromycin, neomycin, hygromycin B, and blasticidin, glutamine synthetase, or dihydrofolate reductase, preferably , the screening marker is human glutamine synthetase.
  4. 根据权利要求2的表达载体，其中，所述筛选标记表达单元的启动子为单纯疱疹病毒胸腺嘧啶激酶启动子、猴空泡病毒40启动子，和/或The expression vector according to claim 2, wherein the promoter of the selection marker expression unit is a herpes simplex virus thymidine kinase promoter, a simian vacuolar virus 40 promoter, and/or
    所述筛选标记表达单元的终止子为猴空泡病毒40聚腺苷化信号，牛生长激素聚腺苷化信号，单纯疱疹病毒胸腺嘧啶激酶聚腺苷化信号或兔β-珠蛋白聚腺苷化信号，优选为猴空泡病毒40聚腺苷化信号；The terminator of the selection marker expression unit is the polyadenylation signal of simian vacuolar virus 40, the polyadenylation signal of bovine growth hormone, the polyadenylation signal of herpes simplex virus thymidine kinase or the polyadenylation signal of rabbit β-globin A Y signal, preferably a simian vacuolar virus 40 polyadenylation signal;
    优选地，所述筛选标记表达单元包括单纯疱疹病毒胸腺嘧啶激酶启动子的编码序列和猴空泡病毒40聚腺苷化信号的编码序列；Preferably, the selection marker expression unit includes the coding sequence of the herpes simplex virus thymidine kinase promoter and the coding sequence of the simian vacuolar virus 40 polyadenylation signal;
    优选地，所述筛选标记表达单元包括单纯疱疹病毒胸腺嘧啶激酶启动子的编码序列，人谷氨酰胺合成酶筛选标记的编码序列和猴空泡病毒40聚腺苷化信号的编码序列。Preferably, the selection marker expression unit includes the coding sequence of herpes simplex virus thymidine kinase promoter, the coding sequence of human glutamine synthetase screening marker and the coding sequence of simian vacuolar virus 40 polyadenylation signal.
  5. 根据权利要求1的表达载体，其中，所述目的基因表达单元包括目的基因序列，以及与目的基因序列可操作连接的第二调控序列，The expression vector according to claim 1, wherein the target gene expression unit comprises a target gene sequence, and a second regulatory sequence operably linked to the target gene sequence,
    优选地，所述第二调控序列包括目的基因表达调控元件的编码序列和终止子的编码序列；Preferably, the second regulatory sequence includes the coding sequence of the target gene expression regulatory element and the coding sequence of the terminator;
    优选地，所述目的基因表达调控元件的编码序列具有选自SEQ ID NO.1-6所示的序列；Preferably, the coding sequence of the target gene expression regulatory element has a sequence selected from the sequences shown in SEQ ID NO.1-6;
    优选地，所述目的基因表达单元的终止子为猴空泡病毒40聚腺苷化信号，牛生长激素聚腺苷化信号，单纯疱疹病毒胸腺嘧啶激酶聚腺苷化信号或兔β-珠蛋白聚腺苷化信号，优选为牛生长激素聚腺苷化信号。Preferably, the terminator of the target gene expression unit is a simian vacuolar virus 40 polyadenylation signal, a bovine growth hormone polyadenylation signal, a herpes simplex virus thymidine kinase polyadenylation signal or a rabbit β-globin The polyadenylation signal, preferably the bovine growth hormone polyadenylation signal.
  6. 一种表达系统，所述表达系统包括权利要求1-5任一项的表达载体。An expression system comprising the expression vector according to any one of claims 1-5.
  7. 根据权利要求6的表达系统，还包含编码PiggyBac转座酶的辅助质粒。The expression system according to claim 6, further comprising a helper plasmid encoding PiggyBac transposase.
  8. 一种宿主细胞，所述宿主细胞转染有权利要求1-5任一项的表达载体，或权利要求6 或7的表达系统，A host cell transfected with the expression vector of any one of claims 1-5, or the expression system of claim 6 or 7,
    优选地，所述宿主细胞的基因组中稳定整合有权利要求1-5任一项的表达载体；Preferably, the expression vector according to any one of claims 1-5 is stably integrated in the genome of the host cell;
    优选地，所述宿主细胞不包含编码PiggyBac转座酶的辅助质粒；Preferably, the host cell does not contain a helper plasmid encoding PiggyBac transposase;
    优选地，所述宿主细胞为CHO细胞。Preferably, the host cells are CHO cells.
  9. 利用权利要求1-5任一项的表达载体或权利要求6或7的表达系统筛选细胞的方法，所述方法包括以下步骤：A method for screening cells using the expression vector of any one of claims 1-5 or the expression system of claim 6 or 7, said method comprising the following steps:
    (a)用权利要求1-5任一项的表达载体或权利要求6或7的表达系统转染宿主细胞；以及(a) transfect host cells with the expression vector of any one of claims 1-5 or the expression system of claim 6 or 7; and
    (b)利用针对所述表达载体中的筛选标记的筛选试剂对步骤(a)获得的宿主细胞进行筛选的步骤。(b) a step of screening the host cells obtained in step (a) with a screening reagent directed against the screening marker in the expression vector.
  10. 根据权利要求9的方法，其中，所述宿主细胞为CHO细胞；The method according to claim 9, wherein the host cell is a CHO cell;
    优选地，所述筛选标记为嘌呤霉素，并且使用10μg/ml的嘌呤霉素对宿主细胞进行筛选，所述宿主细胞转染有包含嘌呤霉素抗性筛选标记的所述表达载体；Preferably, the selection marker is puromycin, and 10 μg/ml of puromycin is used to select host cells transfected with the expression vector comprising a puromycin resistance selection marker;
    优选地，所述筛选标记为嘌呤霉素，并且使用10μg/ml、50μg/ml、250μg/ml浓度增高的嘌呤霉素对宿主细胞进行筛选，所述宿主细胞转染有包含嘌呤霉素抗性筛选标记的所述表达载体；Preferably, the screening marker is puromycin, and the host cells are screened using puromycin with increased concentrations of 10 μg/ml, 50 μg/ml, and 250 μg/ml, and the host cells are transfected with The expression vector of the screening marker;
    优选地，所述筛选标记为谷氨酰胺合成酶，并且使用25μM的蛋氨酸亚氨基代砜对宿主细胞进行筛选，所述宿主细胞转染有包含谷氨酰胺合成酶筛选标记的所述表达载体；Preferably, the screening marker is glutamine synthetase, and 25 μM methionine iminosulfone is used to screen the host cells transfected with the expression vector comprising the glutamine synthetase screening marker;
    优选地，所述筛选标记为谷氨酰胺合成酶，并且使用25μM、50μM浓度增高的蛋氨酸亚氨基代砜对宿主细胞进行筛选，所述宿主细胞转染有包含谷氨酰胺合成酶筛选标记的所述表达载体；Preferably, the screening marker is glutamine synthetase, and the host cells are screened using 25 μM, 50 μM methionine iminosulfone at increased concentrations, and the host cells are transfected with the glutamine synthetase screening marker. expression vector;
    优选地，所述筛选标记为谷氨酰胺合成酶筛选标记，并且使用25μM、50μM、100μM浓度增高的蛋氨酸亚氨基代砜对宿主细胞进行筛选，所述宿主细胞转染有谷氨酰胺合成酶筛选标记的所述表达载体；Preferably, the screening marker is a glutamine synthetase screening marker, and the host cells are screened using 25 μM, 50 μM, and 100 μM of methionine iminosulfone with increased concentrations, and the host cells are transfected with glutamine synthetase screening The expression vector of the mark;
    优选地，所述筛选标记为人谷氨酰胺合成酶；Preferably, the screening marker is human glutamine synthetase;
    优选地，所述筛选标记为二氢叶酸还原酶，并且使用250nM的氨甲喋呤对宿主细胞进行筛选，所述宿主细胞转染有包含二氢叶酸还原酶筛选标记的所述表达载体；Preferably, the screening marker is dihydrofolate reductase, and 250 nM methotrexate is used to screen host cells transfected with the expression vector comprising the dihydrofolate reductase screening marker;
    优选地，所述筛选标记为二氢叶酸还原酶，并且使用250nM、500nM浓度增高的氨甲喋呤对宿主细胞进行筛选，所述宿主细胞转染有包含二氢叶酸还原酶筛选标记的所述表达载体。Preferably, the screening marker is dihydrofolate reductase, and methotrexate with increased concentration of 250 nM and 500 nM is used to screen host cells, and the host cells are transfected with the expression vector containing dihydrofolate reductase screening marker.
  11. 由权利要求10的筛选方法获得的细胞。Cells obtained by the screening method of claim 10.
  12. 权利要求1-5任一项的表达载体，权利要求6或7的表达系统，权利要求8的宿主细胞，或权利要求9或10的筛选方法，或权利要求11的细胞在生产表达量提高的目的蛋白中的应用。The expression vector according to any one of claims 1-5, the expression system according to claim 6 or 7, the host cell according to claim 8, or the screening method according to claim 9 or 10, or the cell according to claim 11 in the production of increased expression application in the target protein.
PCT/CN2023/071371 2022-01-10 2023-01-09 Expression vector and use thereof WO2023131330A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210022828.1A CN116445542A (en) 2022-01-10 2022-01-10 Expression vector and application thereof
CN202210022828.1 2022-01-10

Publications (1)

Publication Number Publication Date
WO2023131330A1 true WO2023131330A1 (en) 2023-07-13

Family

ID=87073307

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/071371 WO2023131330A1 (en) 2022-01-10 2023-01-09 Expression vector and use thereof

Country Status (2)

Country Link
CN (1) CN116445542A (en)
WO (1) WO2023131330A1 (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090042297A1 (en) * 2007-06-01 2009-02-12 George Jr Alfred L Piggybac transposon-based vectors and methods of nucleic acid integration
CN106755096A (en) * 2016-12-20 2017-05-31 上海药明生物技术有限公司 The method for obtaining the stable cell mass of expression target protein in Chinese hamster ovary celI using piggyBac transposon
WO2020068631A1 (en) * 2018-09-24 2020-04-02 Merck Sharp & Dohme Corp. Expression vectors for eukaryotic expression systems
WO2020132382A1 (en) * 2018-12-21 2020-06-25 Merck Sharp & Dohme Corp. Expression vectors for eukaryotic expression systems
US20200318121A1 (en) * 2019-04-08 2020-10-08 Dna Twopointo Inc. Integration of nucleic acid constructs into eukaryotic cells with a transposase from oryzias
CN112513277A (en) * 2019-04-08 2021-03-16 Dna2.0股份有限公司 Transposition of nucleic acid constructs into eukaryotic genomes using transposase from cartap
WO2021164704A2 (en) * 2020-02-19 2021-08-26 Wuxi Biologics (Shanghai) Co., Ltd. Enhanced expression system and methods of use thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090042297A1 (en) * 2007-06-01 2009-02-12 George Jr Alfred L Piggybac transposon-based vectors and methods of nucleic acid integration
CN106755096A (en) * 2016-12-20 2017-05-31 上海药明生物技术有限公司 The method for obtaining the stable cell mass of expression target protein in Chinese hamster ovary celI using piggyBac transposon
WO2020068631A1 (en) * 2018-09-24 2020-04-02 Merck Sharp & Dohme Corp. Expression vectors for eukaryotic expression systems
WO2020132382A1 (en) * 2018-12-21 2020-06-25 Merck Sharp & Dohme Corp. Expression vectors for eukaryotic expression systems
US20200318121A1 (en) * 2019-04-08 2020-10-08 Dna Twopointo Inc. Integration of nucleic acid constructs into eukaryotic cells with a transposase from oryzias
CN112424362A (en) * 2019-04-08 2021-02-26 Dna2.0股份有限公司 Integration of a nucleic acid construct into a eukaryotic cell using transposase from medaka
CN112513277A (en) * 2019-04-08 2021-03-16 Dna2.0股份有限公司 Transposition of nucleic acid constructs into eukaryotic genomes using transposase from cartap
WO2021164704A2 (en) * 2020-02-19 2021-08-26 Wuxi Biologics (Shanghai) Co., Ltd. Enhanced expression system and methods of use thereof

Also Published As

Publication number Publication date
CN116445542A (en) 2023-07-18

Similar Documents

Publication Publication Date Title
Benton et al. The use of UCOE vectors in combination with a preadapted serum free, suspension cell line allows for rapid production of large quantities of protein
CN102648288B (en) Inhibition-based high-throughput screen strategy for cell clones
CN102197136B (en) Cell surface display by the polypeptide isoforms of termination codon liaison
EP3075858B1 (en) Mammalian expression vector
JP4660046B2 (en) Vectors and methods for recombinant protein expression
WO2008121324A2 (en) Recombinant expression vector elements (reves) for enhancing expression of recombinant proteins in host cells
US11807855B2 (en) Selection vectors and methods of selecting eukaryotic host cells
US20130157312A1 (en) Novel intergenic elements for enhancing gene expression
JPWO2012081628A1 (en) Protein production method
US20130273650A1 (en) Vectors and methods for recombinant protein expression
WO2023131330A1 (en) Expression vector and use thereof
Shen et al. Recombinant DNA technology and cell line development
EP2367943B1 (en) Mammalian expression vector
US7674618B2 (en) Expression vector
JP5745431B2 (en) Method for selecting eukaryotic cells expressing heterologous proteins
AU2005306356A1 (en) Methods for producing mammalian cells
TW201827598A (en) Early post-transfection isolation of cells (epic) for biologics production
Xiong et al. Expression of chimeric antibody in mammalian cells using dicistronic expression vector
WO2023246626A1 (en) Cell with targeted integration, method for preparing same, and method for producing target gene expression product
EP2638146A1 (en) Nuclear dna content of cho cell lines as a selection criterion in screening of the best recombinant protein producers
CN117947069A (en) Stable and high-yield targeted integrated cell, and preparation method and application thereof
SE543999C2 (en) Dna construct for expressing a novel signal peptide
SE2030085A1 (en) Dna construct for expressing nivolumab
SE544001C2 (en) Novel combination of tis sequences, signal peptide sequences and nucleic acid sequences encoding heavy and light chains of an antibody
SE544000C2 (en) Novel combination of tis sequence, signal peptide sequence and nucleic acid sequence encoding a recombinant protein

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23737192

Country of ref document: EP

Kind code of ref document: A1