WO2023246626A1 - Cell with targeted integration, method for preparing same, and method for producing target gene expression product - Google Patents
Cell with targeted integration, method for preparing same, and method for producing target gene expression product Download PDFInfo
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- WO2023246626A1 WO2023246626A1 PCT/CN2023/100580 CN2023100580W WO2023246626A1 WO 2023246626 A1 WO2023246626 A1 WO 2023246626A1 CN 2023100580 W CN2023100580 W CN 2023100580W WO 2023246626 A1 WO2023246626 A1 WO 2023246626A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- 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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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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
- C12N15/67—General methods for enhancing the expression
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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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12N2510/00—Genetically modified cells
Definitions
- the present invention relates to the field of biotechnology, and in particular to targeted integration cells and preparation methods thereof, as well as methods for producing target gene expression products.
- the traditional cell line construction strategy mainly involves randomly integrating nucleotide sequences encoding target polypeptides. This method has several shortcomings, including:
- the target gene In terms of the construction process, after the target gene is transfected into CHO cells, after a period of pressure screening, the target gene will gradually be integrated into the host cell genome. Due to the low integration frequency and strong randomness, the resulting screened population (i.e., cells pool) exhibit complex diversity that may result in a wide variety of gene expression and cell growth phenotypes, termed "position-effect variation.” Moreover, a large number of monoclonal blind selections and amplifications are required to finalize high-yield and stable cell lines. This method requires a lot of screening work for each cell pool, and subsequent process development and culture for each cell line is required. Basic optimization consumes a lot of repetitive work, resulting in a waste of resources and high costs.
- the expression stability of cell lines obtained based on the traditional random integration strategy is difficult to predict during the culture process (especially in the later stages of culture).
- the information of random integration sites is unclear, and the site effect of exogenous target gene integration can also cause a significant decrease in the expression level of the target gene.
- high-stringency selection systems such as the GS (glutamine synthetase) gene expression system to enrich high-producing cell lines can produce gene amplification and increase gene copy number, it sometimes results in unstable cell growth and/or product expression.
- targeted integration cell sites that stably and highly express the target gene
- integrating the target gene into targeted integration cells can improve the controllability and repeatability of the integration process, facilitate stable and high-yield expression of the target gene, and simplify subsequent follow-up Screening and process development steps. Therefore, it is necessary to develop a targeted integration cell for site-specific integration of foreign nucleotide sequences.
- the purpose of the present invention includes providing a targeted integration cell, which contains the nucleic acid fragment shown in SEQ ID No. 1 and integrates an exogenous nucleotide sequence within the nucleic acid fragment.
- a nucleic acid comprising the nucleic acid fragment shown in SEQ ID No. 1, and the exogenous nucleic acid fragment is integrated into the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 100-3000th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 150-2820th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 324-2688th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 480-2460th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 700-2200th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 976-1900th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1260-1879th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1561st-1800th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1678-1744th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1701-1720th base interval of the nucleic acid fragment.
- the exogenous nucleic acid fragment includes: a first recombination recognition sequence and a second recombination recognition sequence recognized by a recombinase, located between the first recombination recognition sequence and the second recombination recognition sequence. selection between marker genes and target genes, as well as regulation The selectable marker gene and the promoter for expression of the target gene.
- the recombinase is Bxb1 integrase, ⁇ C31 integrase, Cre recombinase or FLP recombinase.
- the first recombination recognition sequence and the second recombination recognition sequence are independently selected from one or more of the following sequences: LoxP sequence, LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence , Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1attP sequence, Bxb1attB sequence, attP sequence and attB sequence.
- the selectable marker gene is selected from the group consisting of neomycin resistance gene, thymidine kinase gene, hygromycin phosphotransferase gene, dihydrofolate reductase gene, thymidine kinase gene, glutamine
- neomycin resistance gene thymidine kinase gene
- hygromycin phosphotransferase gene dihydrofolate reductase gene
- thymidine kinase gene glutamine
- the amide synthase gene asparagine synthase gene, tryptophan synthase gene, histidinol dehydrogenase gene, aminoglycoside phosphotransferase gene, tryptophan synthase gene and fluorescent protein gene kind.
- the promoter is CMV promoter, SV40 promoter, RSV promoter, ⁇ -globin promoter, UBC promoter, EF1a promoter, ubiquitin promoter, ⁇ -actin promoter , PGK1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, Eif4A1 promoter, Egr1 promoter, FerH promoter, SM22 ⁇ promoter or Endothelin-1 promoter.
- the target gene encodes one or more of antibodies, recombinant proteins, polypeptides, enzymes, hormones, growth factors and receptors.
- a recombinant vector comprising:
- the recombinant vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, or a papillomavirus vector.
- a targeted integrating cell comprising the nucleic acid described in the first aspect.
- the targeted integration cell is a eukaryotic cell.
- the eukaryotic cells are mammalian cells.
- the targeted integration cells include Chinese hamster ovary CHO cells and human embryonic kidney HEK293 cells.
- a method for preparing targeted integration cells comprising the following steps:
- the nucleic acid described in the first aspect is introduced into a cell, or a cell containing the nucleic acid fragment shown in SEQ ID No. 1 is provided and the exogenous nucleic acid fragment is integrated into the nucleic acid fragment to prepare a targeted integrated cell.
- a method for producing a target gene expression product includes the following steps: cultivating the targeted integration cells described in the third aspect, and collecting the target gene expression product.
- the present invention has the following beneficial effects:
- nucleic acid fragment corresponding to SEQ ID No. 1 or the homologous fragment that maintains at least 90% consistency with it was integrated into the exogenous nucleotide sequence (including target genes, etc.) Within the nucleic acid fragment or its homologous fragment, the corresponding targeted integrated cells are stable and highly productive.
- the targeted integrated cells of the present invention do not have high requirements for subsequent culture processes (such as culture conditions, culture medium formula).
- the present invention provides targeted integration cells, and the exogenous nucleotide sequence insertion site is determined. Therefore, compared with the traditional random integration cell construction method, the construction process required for the targeted integration cells of the present invention is simple, time-consuming, and efficient. Improvement, cost reduction, reduced uncertainty, good repeatability, and strong controllability.
- Figure 1 is an operation flow chart of Embodiment 1;
- Figure 2 is a schematic diagram of RMCE used for site screening in Example 1;
- Figure 3 is a recombinant plasmid map used for site screening in Example 1;
- Figure 4 is a graph showing the growth stability results of GBB-01 fluorescent mother cells
- Figure 5 is a schematic diagram of RMCE used for target gene transfection in Example 1;
- Figure 6 is a recombinant plasmid map used for target gene transfection in Example 1;
- Figure 7 is a diagram showing the evaluation results of the expression level of the target gene fed-batch culture of the targeted integration cells prepared in Example 1;
- Figure 8 is a diagram showing the results of evaluating the stability of the expression level of the targeted integrated cells prepared in Example 1;
- Figure 9 is a diagram showing the results of stability evaluation of the specific yield of targeted integrated cells prepared in Example 1.
- Figure 10 is a comparison chart of the sugar profile of the targeted integrated cells prepared in Example 1 and the sugar profile of the randomly integrated control cell product;
- Figure 11 is a comparison chart of the charge heterogeneity of the products of targeted integration cells and random integration control cells prepared in Example 1;
- Figure 12 is a verification diagram of the integration site of the targeted integration cells prepared in Example 1.
- first”, “second”, “third” and “fourth” etc. are for descriptive purposes only and shall not be understood as indicating or implying relative importance or quantity, nor shall they be understood as implicitly indicating the importance or quantity of indicated technical features.
- first”, “second”, “third”, “fourth”, etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation of quantity.
- the technical features described in open terms include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
- the optional numerical distribution is considered to be continuous within the above-mentioned numerical intervals, and includes the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value). value), and every value between the two numeric endpoints.
- a numerical interval only points to integers within the numerical interval, including the two endpoint integers of the numerical range, and every integer between the two endpoints, in this article, it is equivalent to directly enumerating each Integer, for example, t is an integer selected from 1-10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.
- “about” or “approximately” means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, ie, the limitations of the measurement system. .
- “about” may mean within 3 or more standard deviations, according to practice in the art.
- “about” may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value.
- the term may mean within an order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold.
- a "selectable marker gene” may be a gene that allows targeted integrated cells carrying the gene to be specifically selected for or against the gene in the presence of a corresponding selection agent.
- a selectable marker may allow targeted integrated cells transformed with the selectable marker gene to be positively selected in the presence of the gene; non-transformed targeted integrated cells will be unable to grow or survive under the selection conditions.
- Selectable markers can be positive, negative, or bifunctional. Positive selection markers may allow selection of marker-bearing cells, whereas negative selection markers may allow marker-bearing cells to be selectively eliminated. Selectable markers can confer resistance to drugs or compensate for metabolic or catabolic defects in targeted integrated cells.
- antibody is used in its broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), half-antibodies, and antibodies fragments, so long as the fragments exhibit the desired antigen-binding activity.
- antibody fragment refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds the antigen to which the intact antibody binds.
- antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. sexual antibodies.
- Fv fragment antigen
- Fab fragment antigen binding protein
- Fab' fragment antigen binding protein
- Fab'-SH fragment antigen binding protein 2
- diabodies linear antibodies
- single chain antibody molecules e.g., scFv
- multispecific antibodies formed from antibody fragments sexual antibodies.
- targeted integrating cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Targeted integrated cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom, regardless of passage number.
- the nucleic acid content of the offspring may not be exactly the same as that of the parent cells, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as that screened or selected for in the originally transformed cell.
- the term "vector” refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked.
- the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a targeted integrating cell into which they have been introduced.
- a vector directs expression of a nucleic acid operably linked to it. Such vectors are referred to herein as "expression vectors.”
- homologous fragments refers to sequence fragments that share significant sequence similarity as determined by sequence alignment.
- two sequence fragments may be about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% homologous.
- Alignments are performed using algorithms and computer programs (including but not limited to BLAST, FASTA, and HMME) that compare sequence fragments and are based on factors such as sequence length, sequence identity and similarity, and sequence mismatches and gaps. existence and length) to calculate the statistical significance of the match; for example, it can be the ratio of the length of the similar sequence fragment to the length of the aligned region.
- Homologous sequence fragments can refer to both DNA and protein sequences.
- the targeted integrating cell comprises an exogenous nucleotide sequence integrated at an integration site on the genome of the host cell.
- An "integration site” encompasses the targeted integration of a nucleic acid sequence within the genome of a cell into which a foreign nucleotide sequence is inserted.
- the integration site is between two adjacent nucleotides on the genome of the cell targeted for integration.
- the integration site includes stretches of nucleotides between which foreign nucleotide sequences can be inserted.
- the present invention provides a nucleic acid, which comprises the nucleic acid fragment shown in SEQ ID No. 1, and an exogenous nucleic acid fragment is integrated into the nucleic acid fragment.
- the sequence fragment into which the exogenous nucleic acid fragment is integrated maintains no less than 90% identity with the nucleic acid fragment shown in SEQ ID No. 1, such as 90%, 90.5% , 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99 %, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% consistency.
- the integration site of the exogenous nucleic acid fragment can be the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 100-3000th base range of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 150-2820th base range of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 324-2688th base range of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 480-2460th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 700-2200th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 976-1900th base range of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1260-1879th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1561st-1800th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1678-1744th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1701-1720th base interval of the nucleic acid fragment.
- the integration site of the exogenous nucleic acid fragment corresponds to between the 1711th and 1712th base of the nucleic acid fragment.
- the exogenous nucleic acid fragment includes: a first recombination recognition sequence and a second recombination recognition sequence recognized by a recombinase, located between the first recombination recognition sequence and the second recombination recognition sequence. between the selectable marker gene and the target gene, and the promoter that regulates the expression of the selectable marker gene and the target gene.
- the recombinase is Bxb1 integrase, ⁇ C31 integrase, Cre recombinase or FLP recombinase.
- the first recombination recognition sequence and the second recombination recognition sequence are independently selected from one or more of the following sequences: the recombination recognition sequence is a LoxP sequence, a LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1attP sequence, Bxb1attB sequence, attP sequence and attB sequence.
- the selectable marker gene is selected from the group consisting of neomycin resistance gene, thymidine kinase gene, hygromycin phosphotransferase gene, dihydrofolate reductase gene, thymidine kinase gene, glutamine
- neomycin resistance gene thymidine kinase gene
- hygromycin phosphotransferase gene dihydrofolate reductase gene
- thymidine kinase gene glutamine
- the amide synthase gene asparagine synthase gene, tryptophan synthase gene, histidinol dehydrogenase gene, aminoglycoside phosphotransferase gene, tryptophan synthase gene and fluorescent protein gene kind.
- the promoter is CMV promoter, SV40 promoter, RSV promoter, ⁇ -globin promoter, UBC promoter, EF1a promoter, ubiquitin promoter, ⁇ -actin promoter , PGK1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, Eif4A1 promoter, Egr1 promoter, FerH promoter, SM22 ⁇ promoter or Endothelin-1 promoter.
- the target gene encodes one or more of antibodies, recombinant proteins, polypeptides, enzymes, hormones, growth factors and receptors.
- the invention provides a recombinant vector, which includes:
- the 5’ homology arm that is homologous to the sequence fragment present in the nucleic acid fragment shown in SEQ ID No. 1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment,
- the 3’ homology arm that is homologous to the sequence fragment present in the nucleic acid fragment shown in SEQ ID No. 1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment.
- the recombinant vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, or a papillomavirus vector.
- the present invention provides a targeted integrating cell, which contains the nucleic acid described in the first aspect.
- the targeted integration cell is a eukaryotic cell.
- the eukaryotic cells are mammalian cells.
- the mammalian cells include Chinese hamster ovary CHO cells and human embryonic kidney HEK293 cells.
- the CHO cells include CHO host cells, CHO K1 host cells, CHO K1 SV host cells, DG44 host cells, DUKXB-11 host cells, CHOK1S host cells or CHO K1M host cells.
- the present invention provides a method for preparing targeted integrated cells according to the third aspect, which method includes the following steps:
- nucleic acid described in the first aspect into the cell, or provide a cell containing the nucleic acid fragment shown in SEQ ID No. 1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment and use the exogenous nucleic acid fragment Integrate into the nucleic acid fragment shown in SEQ ID No. 1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment to prepare targeted integration cells.
- the integration method includes but is not limited to site-specific recombination technology derived from homologous recombination technology, which relies on integrase targeting specific recognition sites to achieve gene replacement between the genome and exogenous DNA.
- Genetic engineering operations such as gene knockout and knock-in can be, for example, recombinase-mediated cassette exchange, or CRISPR/Cas9-mediated gene targeted integration.
- the present invention provides a method for producing a target gene expression product, which method includes the following steps: cultivating the targeted integration cells described in the third aspect, and collecting the target gene expression product.
- the targeted integrated cells according to this method are stable and highly productive.
- the integration site and/or the nucleotide sequences flanking the integration site can be identified experimentally: In some embodiments of the invention, the integration site can be identified by genome-wide screening methods nucleotide sequences that point and/or flank the integration site to isolate the host cell; in some embodiments of the invention, this can be identified by genome-wide screening methods following a transposase-based cassette integration event.
- nucleotide sequences that identify the integration site and/or flank the integration site; in some embodiments of the invention, the integration site and/or flanking integration site can be identified by brute force random integration screening
- the nucleotide sequence; in some embodiments of the invention, the integration site and/or the flanking integration site can be determined by conventional sequencing methods (such as target locus amplification), followed by next-generation sequencing and whole genome
- the nucleotide sequence; in some embodiments of the invention, the location of the integration site on the chromosome can be determined by conventional cell biology methods (such as fluorescence in situ hybridization analysis).
- the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
- the consumables in the following specific examples involve: Neon Resuspension Buffer R (ThermoFisher), a special resuspension buffer for cell electroporation apparatus, electroporation solution E (ThermoFisher), recovery medium EX-CELLAdvanced CHO Fed-batch Medium (Sigma-Aldrich) , the amplification medium is EX-CELLAdvanced CHO Fed-batch Medium plus 200 ⁇ g/ml hygromycin (ThermoFisher) and 1% GlutaMAX (ThermoFisher), the shake flask medium is EX-CELLAdvanced CHO Fed-batch Medium plus 1% GlutaMAX, fed-batch
- the basal medium in the culture medium uses 100% EX-CELLAdvanced CHO Fed-batch Medium, and the feed medium is 4% Cell Boost 7a/7b (HyClone).
- step (6) Take 15 ⁇ g of the linearized plasmid and resuspend the cells in step (5), mix gently, and pipet 50 times to avoid bubbles;
- step (6) Inhale the cell plasmid suspension in step (6) into the electroporation gun tip, put it into the electroshock tank, and perform electroshock;
- step (1) clone the target gene (Fc fusion protein) fragment into the plasmid, and construct a recombinant plasmid containing RMCE ( Figure 5), see Figure 6;
- the target gene fragment has the sequence shown in SEQ ID No. 2, SEQ ID No. 2:
- step (14) Select the stable high-fluorescence cell GBB-01 from step (14) for transfection and integration verification. After amplifying and linearizing the recombinant plasmid from step (15) above, co-transfect it with Bxb-1 integrase into the stable high-fluorescence cell GBB-01. For cells GBB-01, the transfection steps were as described above;
- the cell pool was subjected to pressure screening 2 days after transfection, and a stable cell pool was formed in about 2 weeks;
- NC_048596 sequence fragment after integrating the target gene is shown in SEQ ID No.3, SEQ ID No.3:
- the amplified bands were cut and purified, and multiple pairs of primers were designed for DNA sequencing.
- the sequence after splicing of the sequencing results is as follows, which is consistent with the theoretical sequence (sequence SEQ ID No. 3), thus confirming that the target gene is correctly integrated. into the target site.
- the reaction system is 5 ⁇ L of forward and reverse oligo, 5 ⁇ L of 10 ⁇ NEB buffer 2, and make up to 50 ⁇ L with water.
- the system was incubated at 95°C (temperature measurement must be used) for 5 minutes, and then naturally cooled to 25°C.
- the obtained product plasmid was ligated with the pDonor CRISPR/Cas9 vector digested by PvuI.
- the ligation product was transformed into DH5 ⁇ competent cells and spread on an ampicillin-resistant LB solid plate. The next day, single clones were picked and the plasmid was extracted. Finally, the correct Cas9 encoding plasmid was verified through first-generation sequencing.
- the donor plasmid consists of a 5' homology arm, an anti-PD1 monoclonal antibody coding gene, a hygromycin coding gene and a 3' homology arm connected in series on the pUC19 plasmid.
- adding Cas9 encoding plasmid and anti-PD1 donor plasmid for co-transfection can obtain a site-directed integrated cell pool, and only adding anti-PD1 donor plasmid for transfection can obtain a randomly integrated cell pool expressing anti-PD1.
- 5’/3’junction PCR is used to initially identify the site-specific integrated cell pool, and the amplified target fragment is sequenced. After confirming that the site-directed integrated cell pool is obtained, the supernatant is collected.
- Obtain monoclonal cell lines by limiting dilution method Use the previous site-specific integrated cell pool to obtain monoclonal cell lines by limiting dilution method. That is, the cell pool was spread on a 96-well plate at a cell density of 0.8 cells/well. Check the board on the same day to determine the single clone. When the cells grow for about 5 weeks, aspirate 1 ⁇ 10 6 monoclonal cells and extract genomic DNA, and perform 5'/3'junction PCR detection again, using the same method as step (5). The positive cell lines detected by Junction PCR were further tested by Western blot to determine whether the anti-PD1 antibody was correctly expressed.
- Targeted integration cells were constructed according to the steps of Example 1.
- the cell lines with single copy random integration were used as controls.
- the expression levels of 6-well plate batch culture were shown in Table 2.
- the cell lines with single copy random integration anti -The expression level of PD1 antibody in 6-well plate batch culture is 285 mg/L, while the expression level of the targeted integration cell line is more than 2 times that level.
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Abstract
Provided are a cell with targeted integration, a method for preparing same, and a method for producing a target gene expression product. The cell with targeted integration comprises a nucleic acid fragment set forth in SEQ ID NO. 1 comprising an integrated exogenous nucleotide sequence. By means of massive screenings in the early stage, the nucleic acid fragment set forth in SEQ ID NO. 1 was found. By integrating the exogenous nucleotide sequence into the nucleic acid fragment, the obtained cell with targeted integration features stability and a high yield.
Description
本发明涉及生物技术领域，特别是涉及靶向整合细胞及其制备方法、生产目标基因表达产物的方法。The present invention relates to the field of biotechnology, and in particular to targeted integration cells and preparation methods thereof, as well as methods for producing target gene expression products.
随着生物制药产业的不断发展，对产物表达量及表达稳定性的要求逐渐增高，而细胞系构建是决定产物表达产量及表达稳定性的关键因素。With the continuous development of the biopharmaceutical industry, the requirements for product expression and expression stability are gradually increasing, and cell line construction is a key factor in determining product expression yield and expression stability.
传统细胞系的构建策略主要是随机整合编码目标多肽的核苷酸序列，这种方法具有多个不足之处，包括：The traditional cell line construction strategy mainly involves randomly integrating nucleotide sequences encoding target polypeptides. This method has several shortcomings, including:
从构建工艺方面讲，将目标基因转染入CHO细胞后，经过一段时间的压力筛选，目标基因会逐渐整合到宿主细胞基因组内，由于整合频率较低、随机性强，所得筛选群体(即细胞池)呈现复杂的多样性，可能导致各种各样的基因表达和细胞生长表型，被称为“位置效应变异”。并且，需要通过大量的单克隆盲选、扩增最终确定高产、稳定的细胞株，这种方式对每一个细胞池都需要大量的筛选工作，后续还需针对每一株细胞进行工艺开发和培养基优化，耗费大量的重复性劳动，造成资源的浪费，成本高。In terms of the construction process, after the target gene is transfected into CHO cells, after a period of pressure screening, the target gene will gradually be integrated into the host cell genome. Due to the low integration frequency and strong randomness, the resulting screened population (i.e., cells pool) exhibit complex diversity that may result in a wide variety of gene expression and cell growth phenotypes, termed "position-effect variation." Moreover, a large number of monoclonal blind selections and amplifications are required to finalize high-yield and stable cell lines. This method requires a lot of screening work for each cell pool, and subsequent process development and culture for each cell line is required. Basic optimization consumes a lot of repetitive work, resulting in a waste of resources and high costs.
从制备细胞系质量讲，基于传统随机整合策略获得的细胞系，在培养过程中(特别是培养后期)，其表达稳定性难以预测。并且，随机整合位点的信息不明确，外源目标基因整合的位点效应也会导致目标基因的表达水平显著下降。尽管利用高严谨性选择***例如GS(谷氨酰胺合成酶)基因表达***富集高产细胞系可以产生基因扩增并增加基因拷贝数，但有时会导致不稳定的细胞生长和/或产物表达。In terms of the quality of prepared cell lines, the expression stability of cell lines obtained based on the traditional random integration strategy is difficult to predict during the culture process (especially in the later stages of culture). Moreover, the information of random integration sites is unclear, and the site effect of exogenous target gene integration can also cause a significant decrease in the expression level of the target gene. Although the use of high-stringency selection systems such as the GS (glutamine synthetase) gene expression system to enrich high-producing cell lines can produce gene amplification and increase gene copy number, it sometimes results in unstable cell growth and/or product expression.
利用稳定且高表达目标基因的靶向整合细胞位点，将目标基因定点整合入靶向整合细胞，能够提升整合过程的可控性和重复性，利于目标基因的稳定高产表达，也能够简化后续筛选及工艺开发等步骤。因此，开发一种外源核苷酸序列定点整合的靶向整合细胞是必要的。Utilizing targeted integration cell sites that stably and highly express the target gene, integrating the target gene into targeted integration cells can improve the controllability and repeatability of the integration process, facilitate stable and high-yield expression of the target gene, and simplify subsequent follow-up Screening and process development steps. Therefore, it is necessary to develop a targeted integration cell for site-specific integration of foreign nucleotide sequences.
发明内容Contents of the invention
基于此，本发明的目的包括提供一种靶向整合细胞，其包含SEQ ID No.1所示的核酸片段且所述核酸片段内整合外源核苷酸序列。Based on this, the purpose of the present invention includes providing a targeted integration cell, which contains the nucleic acid fragment shown in SEQ ID No. 1 and integrates an exogenous nucleotide sequence within the nucleic acid fragment.
本发明的目的可以通过以下技术方案实现：The object of the present invention can be achieved through the following technical solutions:
在本发明的第一方面，提供一种核酸，所述核酸包含SEQ ID No.1所示的核酸片段，所述核酸片段内整合有外源核酸片段。In a first aspect of the present invention, a nucleic acid is provided, the nucleic acid comprising the nucleic acid fragment shown in SEQ ID No. 1, and the exogenous nucleic acid fragment is integrated into the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第100-3000个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 100-3000th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第150-2820个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 150-2820th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第324-2688个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 324-2688th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第480-2460个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 480-2460th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第700-2200个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 700-2200th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第976-1900个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 976-1900th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第1260-1879个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1260-1879th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第1561-1800个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1561st-1800th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第1678-1744个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1678-1744th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段的整合位点对应所述核酸片段的第1701-1720个碱基区间内的任意位点。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1701-1720th base interval of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段包含：重组酶识别的第一重组识别序列和第二重组识别序列，位于所述第一重组识别序列和所述第二重组识别序列之间的选择标记基因和目标基因，以及调控
所述选择标记基因和所述目标基因表达的启动子。In some embodiments of the invention, the exogenous nucleic acid fragment includes: a first recombination recognition sequence and a second recombination recognition sequence recognized by a recombinase, located between the first recombination recognition sequence and the second recombination recognition sequence. selection between marker genes and target genes, as well as regulation The selectable marker gene and the promoter for expression of the target gene.
在本发明的一些实施方式中，所述重组酶为Bxb1整合酶、ΦC31整合酶、Cre重组酶或者FLP重组酶。In some embodiments of the invention, the recombinase is Bxb1 integrase, ΦC31 integrase, Cre recombinase or FLP recombinase.
在本发明的一些实施方式中，所述第一重组识别序列和所述第二重组识别序列独立地选自如下序列中的一种或者多种：LoxP序列、LoxPL3序列、LoxP 2L序列、LoxFas序列、Lox511序列、Lox2272序列、Lox2372序列、Lox5171序列、Loxm2序列、Lox71序列、Lox66序列、FRT序列、Bxb1attP序列、Bxb1attB序列、attP序列和attB序列。In some embodiments of the invention, the first recombination recognition sequence and the second recombination recognition sequence are independently selected from one or more of the following sequences: LoxP sequence, LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence , Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1attP sequence, Bxb1attB sequence, attP sequence and attB sequence.
在本发明的一些实施方式中，所述选择标记基因选自新霉素抗性基因、胸苷激酶基因、潮霉素磷酸转移酶基因、二氢叶酸还原酶基因、胸苷激酶基因、谷氨酰胺合成酶基因、天冬酰胺合成酶基因、色氨酸合成酶基因、组氨醇脱氢酶基因、氨基糖苷磷酸转移酶基因、色氨酸合成酶基因和荧光蛋白基因中的一种或者多种。In some embodiments of the invention, the selectable marker gene is selected from the group consisting of neomycin resistance gene, thymidine kinase gene, hygromycin phosphotransferase gene, dihydrofolate reductase gene, thymidine kinase gene, glutamine One or more of the amide synthase gene, asparagine synthase gene, tryptophan synthase gene, histidinol dehydrogenase gene, aminoglycoside phosphotransferase gene, tryptophan synthase gene and fluorescent protein gene kind.
在本发明的一些实施方式中，所述启动子为CMV启动子、SV40启动子、RSV启动子、β-globin启动子、UBC启动子、EF1a启动子、泛素启动子、β-actin启动子、PGK1启动子、Rosa26启动子、HSP70启动子、GAPDH启动子、Eif4A1启动子、Egr1启动子、FerH启动子、SM22α启动子或者Endothelin-1启动子。In some embodiments of the invention, the promoter is CMV promoter, SV40 promoter, RSV promoter, β-globin promoter, UBC promoter, EF1a promoter, ubiquitin promoter, β-actin promoter , PGK1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, Eif4A1 promoter, Egr1 promoter, FerH promoter, SM22α promoter or Endothelin-1 promoter.
在本发明的一些实施方式中，所述目标基因编码抗体、重组蛋白、多肽、酶、激素、生长因子和受体中的一种或者多种。In some embodiments of the invention, the target gene encodes one or more of antibodies, recombinant proteins, polypeptides, enzymes, hormones, growth factors and receptors.
在本发明的第二方面，提供一种重组载体，所述重组载体包含：In a second aspect of the present invention, a recombinant vector is provided, the recombinant vector comprising:
a.与SEQ ID No.1所示的核酸片段所存在的序列片段同源的5’同源臂，a. The 5’ homology arm that is homologous to the sequence fragment present in the nucleic acid fragment shown in SEQ ID No. 1,
b.目标基因，b. Target gene,
c.与SEQ ID No.1所示的核酸片段所存在的序列片段同源的3’同源臂。c. The 3’ homology arm that is homologous to the sequence fragment present in the nucleic acid fragment shown in SEQ ID No. 1.
在本发明的一些实施方式中，所述重组载体为慢病毒载体、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体、杆状病毒载体、***瘤病毒载体、***多瘤空泡病毒载体、整合性噬菌体载体、非病毒载体、转座子和/或转座酶、整合酶底物或者质粒。In some embodiments of the invention, the recombinant vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, or a papillomavirus vector. Viral vectors, integrating phage vectors, non-viral vectors, transposons and/or transposases, integrase substrates or plasmids.
在本发明的第三方面，提供一种靶向整合细胞，所述靶向整合细胞包含第一方面所述的核酸。In a third aspect of the present invention, a targeted integrating cell is provided, the targeted integrating cell comprising the nucleic acid described in the first aspect.
在本发明的一些实施方式中，所述靶向整合细胞为真核细胞。In some embodiments of the invention, the targeted integration cell is a eukaryotic cell.
在本发明的一些实施方式中，所述真核细胞为哺乳动物细胞。In some embodiments of the invention, the eukaryotic cells are mammalian cells.
在本发明的一些实施方式中，所述靶向整合细胞包括中国仓鼠卵巢CHO细胞、人胚肾HEK293细胞。In some embodiments of the invention, the targeted integration cells include Chinese hamster ovary CHO cells and human embryonic kidney HEK293 cells.
在本发明的第四方面，提供第三方面所述的靶向整合细胞的制备方法，所述制备方法包括如下步骤：In a fourth aspect of the present invention, there is provided a method for preparing targeted integration cells according to the third aspect, the preparation method comprising the following steps:
将第一方面所述的核酸导入细胞，或者提供包含SEQ ID No.1所示的核酸片段的细胞并将所述外源核酸片段整合至所述核酸片段内，制备靶向整合细胞。The nucleic acid described in the first aspect is introduced into a cell, or a cell containing the nucleic acid fragment shown in SEQ ID No. 1 is provided and the exogenous nucleic acid fragment is integrated into the nucleic acid fragment to prepare a targeted integrated cell.
在本发明的第五方面，提供生产目标基因表达产物的方法，所述方法包括如下步骤：培养第三方面所述的靶向整合细胞，收集目标基因表达产物。In a fifth aspect of the present invention, a method for producing a target gene expression product is provided, which method includes the following steps: cultivating the targeted integration cells described in the third aspect, and collecting the target gene expression product.
相对于传统技术，本发明具备如下有益效果：Compared with traditional technology, the present invention has the following beneficial effects:
本发明经过前期大量的筛选，发现对应如SEQ ID No.1所示的核酸片段或者与其保持至少90％一致性的同源片段，将外源核苷酸序列(包括目标基因等)整合到该核酸片段或其同源片段内，对应得到的靶向整合细胞具有稳定且高产的特点。After extensive screening in the early stage, the present invention found that the nucleic acid fragment corresponding to SEQ ID No. 1 or the homologous fragment that maintains at least 90% consistency with it was integrated into the exogenous nucleotide sequence (including target genes, etc.) Within the nucleic acid fragment or its homologous fragment, the corresponding targeted integrated cells are stable and highly productive.
并且，本发明靶向整合细胞基于其稳定、高产的特点，对后续培养工艺(例如培养条件、培养基配方)要求不高。Moreover, based on its stable and high-yield characteristics, the targeted integrated cells of the present invention do not have high requirements for subsequent culture processes (such as culture conditions, culture medium formula).
同时，本发明提供靶向整合细胞，外源核苷酸序列***位点确定，因此相对于传统随机整合的细胞构建方法，本发明靶向整合细胞所需的构建工艺简单，耗时缩短，效率提升，成本降低，并且不确定性降低，重复性好，可控性强。At the same time, the present invention provides targeted integration cells, and the exogenous nucleotide sequence insertion site is determined. Therefore, compared with the traditional random integration cell construction method, the construction process required for the targeted integration cells of the present invention is simple, time-consuming, and efficient. Improvement, cost reduction, reduced uncertainty, good repeatability, and strong controllability.
为了更清楚地说明本发明实施例中的技术方案、更完整地理解本发明及其有益效果，下面将对实施例描述中所需要使用的附图作简单的介绍。显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对本领域技术人员来说，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to explain the technical solutions in the embodiments of the present invention more clearly and to understand the present invention and its beneficial effects more completely, the drawings needed to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exerting creative efforts.
图1为实施例1的操作流程图；
Figure 1 is an operation flow chart of Embodiment 1;
图2为实施例1用于位点筛选的RMCE示意图；Figure 2 is a schematic diagram of RMCE used for site screening in Example 1;
图3为实施例1用于位点筛选的重组质粒图谱；Figure 3 is a recombinant plasmid map used for site screening in Example 1;
图4为GBB-01荧光母细胞生长稳定性结果图；Figure 4 is a graph showing the growth stability results of GBB-01 fluorescent mother cells;
图5为实施例1用于目标基因转染的RMCE示意图；Figure 5 is a schematic diagram of RMCE used for target gene transfection in Example 1;
图6为实施例1用于目标基因转染的重组质粒图谱；Figure 6 is a recombinant plasmid map used for target gene transfection in Example 1;
图7为实施例1制备的靶向整合细胞的目标基因流加培养表达量评估结果图；Figure 7 is a diagram showing the evaluation results of the expression level of the target gene fed-batch culture of the targeted integration cells prepared in Example 1;
图8为实施例1制备的靶向整合细胞表达量稳定性评估结果图；Figure 8 is a diagram showing the results of evaluating the stability of the expression level of the targeted integrated cells prepared in Example 1;
图9为实施例1制备的靶向整合细胞比产率稳定性评估结果图；Figure 9 is a diagram showing the results of stability evaluation of the specific yield of targeted integrated cells prepared in Example 1;
图10为实施例1制备的靶向整合细胞糖谱与随机整合对照细胞产品糖谱对比图；Figure 10 is a comparison chart of the sugar profile of the targeted integrated cells prepared in Example 1 and the sugar profile of the randomly integrated control cell product;
图11为实施例1制备的靶向整合细胞与随机整合对照细胞产品电荷异质性对比图；Figure 11 is a comparison chart of the charge heterogeneity of the products of targeted integration cells and random integration control cells prepared in Example 1;
图12为实施例1制备的靶向整合细胞整合位点验证图。Figure 12 is a verification diagram of the integration site of the targeted integration cells prepared in Example 1.
下面结合附图、实施方式和实施例，对本发明作进一步详细的说明。应理解，这些实施方式和实施例仅用于说明本发明而不用于限制本发明的范围，提供这些实施方式和实施例的目的是使对本发明公开内容理解更加透彻全面。还应理解，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施方式和实施例，本领域技术人员可以在不违背本发明内涵的情况下作各种改动或修改，得到的等价形式同样落于本发明的保护范围。此外，在下文的描述中，给出了大量具体的细节以便提供对本发明更为充分地理解，应理解，本发明可以无需一个或多个这些细节而得以实施。The present invention will be described in further detail below with reference to the drawings, implementation modes and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the implementation modes and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and obtain The equivalent forms also fall within the protection scope of the present invention. Additionally, in the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention, and it is to be understood that the invention may be practiced without one or more of these details.
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述实施方式和实施例的目的，不是旨在于限制本发明。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the invention belongs. The terminology used herein in the description of the invention is for the purpose of describing embodiments and examples only and is not intended to limit the invention.
术语the term
除非另外说明或存在矛盾之处，本文中使用的术语或短语具有以下含义：Unless otherwise stated or contradictory, the terms or phrases used in this article have the following meanings:
本发明中，“进一步”、“更进一步”、“特别”等用于描述目的，表示内容上的差异，但并不应理解为对本发明保护范围的限制。In the present invention, "further", "further", "especially", etc. are used for description purposes and indicate differences in content, but should not be understood as limiting the scope of the present invention.
本发明中，“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“可选”各自独立。In the present invention, "optionally", "optional" and "optional" mean that it is optional, that is, it refers to any one selected from the two parallel solutions of "with" or "without". If there are multiple "optionals" in a technical solution, each "optional" will be independent unless otherwise specified and there is no contradiction or mutual restriction.
本发明中，“第一方面”、“第二方面”、“第三方面”、“第四方面”等中，术语“第一”、“第二”、“第三”、“第四”等仅用于描述目的，不能理解为指示或暗示相对重要性或数量，也不能理解为隐含指明所指示的技术特征的重要性或数量。而且“第一”、“第二”、“第三”、“第四”等仅起到非穷举式的列举描述目的，应当理解并不构成对数量的封闭式限定。In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third" and "fourth" etc. are for descriptive purposes only and shall not be understood as indicating or implying relative importance or quantity, nor shall they be understood as implicitly indicating the importance or quantity of indicated technical features. Furthermore, “first”, “second”, “third”, “fourth”, etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation of quantity.
本发明中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。In the present invention, the technical features described in open terms include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
本发明中，涉及到数值区间(也即数值范围)，如无特别说明，可选的数值分布在上述数值区间内视为连续，且包括该数值范围的两个数值端点(即最小值及最大值)，以及这两个数值端点之间的每一个数值。如无特别说明，当数值区间仅仅指向该数值区间内的整数时，包括该数值范围的两个端点整数，以及两个端点之间的每一个整数，在本文中，相当于直接列举了每一个整数，比如t为选自1-10的整数，表示t为选自由1、2、3、4、5、6、7、8、9和10构成的整数组的任一个整数。此外，当提供多个范围描述特征或特性时，可以合并这些范围。换言之，除非另有指明，否则本文中所公开之范围应理解为包括其中所归入的任何及所有的子范围。In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered to be continuous within the above-mentioned numerical intervals, and includes the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value). value), and every value between the two numeric endpoints. Unless otherwise specified, when a numerical interval only points to integers within the numerical interval, including the two endpoint integers of the numerical range, and every integer between the two endpoints, in this article, it is equivalent to directly enumerating each Integer, for example, t is an integer selected from 1-10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Additionally, when multiple scopes are provided to describe a feature or characteristic, these scopes can be combined. In other words, unless otherwise indicated, the ranges disclosed herein should be understood to include any and all subranges subsumed therein.
在本发明提及的所有文献都在本发明中引用作为参考，就如同每一篇文献被单独引用作为参考那样。除非和本发明的发明目的和/或技术方案相冲突，否则，本发明涉及的引用文献以全部内容、全部目的被引用。本发明中涉及引用文献时，相关技术特征、术语、名词、短语等在引用文献中的定义也一并被引用。本发明中涉及引用文献时，被引用的相关技术特征的举例、优选方式也可作为参考纳入本发明中，但以能够实施本发明为限。应当理解，当引用内容与本发明中的描述相冲突时，以本发明为准或者适应性地根据本发明的描述进行修正。
All documents mentioned herein are herein incorporated by reference to the same extent as if each individual document was individually incorporated by reference. Unless it conflicts with the inventive purpose and/or technical solution of the present invention, the cited documents involved in the present invention are cited in their entirety and for all purposes. When referring to cited documents in the present invention, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When citing documents in the present invention, the cited examples and preferred modes of relevant technical features may also be incorporated into the present invention as references, but only to the extent that the present invention can be implemented. It should be understood that when the quoted content conflicts with the description of the present invention, the present invention shall prevail or be adapted to be modified according to the description of the present invention.
本发明中，“约”或“大约”意指在如通过本领域普通技术人员确定的特定值的可接受误差范围内，其部分取决于如何测量或确定所述值，即测量***的局限性。例如，根据本领域的实践，“约”可以意指在3个或超过3个标准差内。可替代地，“约”可以意指给定值的至多20％、优选至多10％、更优选至多5％、以及又更优选至多1％的范围。可替代地，特别是关于生物***或过程，所述术语可以意指在值的一个数量级内，优选在5倍内，并且更优选在2倍内。As used herein, "about" or "approximately" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, ie, the limitations of the measurement system. . For example, "about" may mean within 3 or more standard deviations, according to practice in the art. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly with regard to biological systems or processes, the term may mean within an order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold.
本发明中，“选择标记基因”可以是如下基因，所述基因允许在存在相应的选择剂的情况下，携带所述基因的靶向整合细胞可对于或针对所述基因而被特异性地选择。例如但非限制性地，选择标记可以允许在存在所述基因的情况下，用选择标记基因转化的靶向整合细胞被阳性选择；非转化的靶向整合细胞将不能在选择条件下生长或存活。选择标记可以是阳性、阴性或双功能的。阳性选择标记可以允许选择携带标记的细胞，而阴性选择标记可以允许携带标记的细胞被选择性地消除。选择标记可以赋予对药物的抗性或补偿靶向整合细胞中的代谢或分解代谢缺陷。In the present invention, a "selectable marker gene" may be a gene that allows targeted integrated cells carrying the gene to be specifically selected for or against the gene in the presence of a corresponding selection agent. . For example, and without limitation, a selectable marker may allow targeted integrated cells transformed with the selectable marker gene to be positively selected in the presence of the gene; non-transformed targeted integrated cells will be unable to grow or survive under the selection conditions. . Selectable markers can be positive, negative, or bifunctional. Positive selection markers may allow selection of marker-bearing cells, whereas negative selection markers may allow marker-bearing cells to be selectively eliminated. Selectable markers can confer resistance to drugs or compensate for metabolic or catabolic defects in targeted integrated cells.
本发明中，“抗体”以最广泛的含义使用，并且包括多种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、半抗体和抗体片段，只要所述片段展现出所需的抗原结合活性。如本文所用，术语“抗体片段”是指除了完整抗体以外的包含完整抗体中结合完整抗体所结合抗原的一部分的分子。抗体片段的例子包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双抗体；线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。有关某些抗体片段的综述，参见Holliger和Hudson,Nature Biotechnology 23:1126-1136(2005)。As used herein, "antibody" is used in its broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), half-antibodies, and antibodies fragments, so long as the fragments exhibit the desired antigen-binding activity. As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. sexual antibodies. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).
本发明中，术语“靶向整合细胞”是指已引入外源核酸的细胞，包括此类细胞的后代。靶向整合细胞包括“转化体”和“转化细胞”，其包括原代转化细胞和源自其的后代，不考虑传代次数。后代的核酸含量可能与亲代细胞不完全相同，但可能含有突变。本文包括具有与针对初始转化细胞中所筛选或选择的相同的功能或生物活性的突变体后代。In the present invention, the term "targeted integrating cell" refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Targeted integrated cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of passage number. The nucleic acid content of the offspring may not be exactly the same as that of the parent cells, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as that screened or selected for in the originally transformed cell.
本发明中，术语“载体”是指能够传播与其连接的另一核酸的核酸分子。所述术语包括作为自我复制核酸结构的载体以及掺入已引入其的靶向整合细胞的基因组中的载体。在某些实施方案中，载体引导与其可操作地连接的核酸的表达。此类载体在本文中称为“表达载体”。In the present invention, the term "vector" refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a targeted integrating cell into which they have been introduced. In certain embodiments, a vector directs expression of a nucleic acid operably linked to it. Such vectors are referred to herein as "expression vectors."
本发明中，术语“同源片段”是指如通过序列比对所确定共有显著序列相似性的序列片段。例如，两个序列片段可以是约50％、60％、70％、80％、90％、95％、99％、或99.9％同源的。比对是通过算法和计算机程序(包括但不限于BLAST、FASTA和HMME)进行的，所述比对比较序列片段并基于因素(如序列长度、序列身份和相似性、以及序列错配和空位的存在和长度)来计算匹配的统计显著性；例如可以是相似序列片段长度与对齐区域长度的比值。同源序列片段可以指DNA和蛋白质序列二者。In the present invention, the term "homologous fragments" refers to sequence fragments that share significant sequence similarity as determined by sequence alignment. For example, two sequence fragments may be about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% homologous. Alignments are performed using algorithms and computer programs (including but not limited to BLAST, FASTA, and HMME) that compare sequence fragments and are based on factors such as sequence length, sequence identity and similarity, and sequence mismatches and gaps. existence and length) to calculate the statistical significance of the match; for example, it can be the ratio of the length of the similar sequence fragment to the length of the aligned region. Homologous sequence fragments can refer to both DNA and protein sequences.
当前公开的主题提供了适合于外源核苷酸序列的靶向整合细胞。在某些实施方案中，靶向整合细胞包含在宿主细胞的基因组上的整合位点处整合的外源核苷酸序列。“整合位点”包含靶向整合细胞基因组内的核酸序列，在所述核酸序列中***外源核苷酸序列。在某些实施方案中，整合位点是在靶向整合细胞基因组上的两个相邻核苷酸之间。在某些实施方案中，整合位点包括核苷酸延伸段，可以在任何所述核苷酸之间***外源核苷酸序列。The presently disclosed subject matter provides cells suitable for targeted integration of exogenous nucleotide sequences. In certain embodiments, the targeted integrating cell comprises an exogenous nucleotide sequence integrated at an integration site on the genome of the host cell. An "integration site" encompasses the targeted integration of a nucleic acid sequence within the genome of a cell into which a foreign nucleotide sequence is inserted. In certain embodiments, the integration site is between two adjacent nucleotides on the genome of the cell targeted for integration. In certain embodiments, the integration site includes stretches of nucleotides between which foreign nucleotide sequences can be inserted.
本发明的第一方面First aspect of the invention
本发明提供一种核酸，所述核酸包含SEQ ID No.1所示的核酸片段，所述核酸片段内整合有外源核酸片段。The present invention provides a nucleic acid, which comprises the nucleic acid fragment shown in SEQ ID No. 1, and an exogenous nucleic acid fragment is integrated into the nucleic acid fragment.
SEQ ID No.1:
SEQ ID No.1:
SEQ ID No.1:
在本发明的一些实施方式中，所述外源核酸片段被整合入的序列片段与所述SEQ ID No.1所示的核酸片段保持不低于90％的一致性，例如90％、90.5％、91％、91.5％、92％、92.5％、93％、93.5％、94％、94.5％、95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％的一致性。In some embodiments of the present invention, the sequence fragment into which the exogenous nucleic acid fragment is integrated maintains no less than 90% identity with the nucleic acid fragment shown in SEQ ID No. 1, such as 90%, 90.5% , 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99 %, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% consistency.
在本发明的一些实施例中，所述外源核酸片段的整合位点可以为所述核酸片段的第1、2、3、4、5、6、7、8、9、10、11、12、……1679、1680、1681、1682、1683、1684、1685、1686、1687、1688、1689、1690、1691、1692、1693、1694、1695、1696、1697、1698、1699、1700、1701、1702、1703、1704、1705、1706、1707、1708、1709、1710、1711、1712、1713、1714、1715、1716、1717、1718、1719、1720、1721、1722、1723、1724、1725、1726、1727、1728、1729、1730、1731、1732、1733、1734、1735、1736、1737、1738、1739、1740、1741、1742、1743、1744……3145、3146、3147、3148、3149、3150个碱基。In some embodiments of the present invention, the integration site of the exogenous nucleic acid fragment can be the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th of the nucleic acid fragment. ,...1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702 ,1703,1704,1705,1706,1707,1708,1709,1710,1711,1712,1713,1714,1715,1716,1717,1718,1719,1720,1721,1722,1723,1724,1725,1726,17 27 ,1728,1729,1730,1731,1732,1733,1734,1735,1736,1737,1738,1739,1740,1741,1742,1743,1744...3145,3146,3147,3148,3149,3150 bases .
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第100-3000个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 100-3000th base range of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第150-2820个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 150-2820th base range of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第324-2688个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 324-2688th base range of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第480-2460个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 480-2460th base interval of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第700-2200个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 700-2200th base interval of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第976-1900个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 976-1900th base range of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第1260-1879个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1260-1879th base interval of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第1561-1800个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1561st-1800th base interval of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第1678-1744个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1678-1744th base interval of the nucleic acid fragment.
可选地，所述外源核酸片段的整合位点对应所述核酸片段的第1701-1720个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1701-1720th base interval of the nucleic acid fragment.
优选地，所述外源核酸片段的整合位点对应所述核酸片段的第1711个和第1712个碱基之间。Preferably, the integration site of the exogenous nucleic acid fragment corresponds to between the 1711th and 1712th base of the nucleic acid fragment.
在本发明的一些实施方式中，所述外源核酸片段包含：重组酶识别的第一重组识别序列和第二重组识别序列，位于所述第一重组识别序列和所述第二重组识别序列之间的选择标记基因和目标基因，以及调控所述选择标记基因和所述目标基因表达的启动子。In some embodiments of the invention, the exogenous nucleic acid fragment includes: a first recombination recognition sequence and a second recombination recognition sequence recognized by a recombinase, located between the first recombination recognition sequence and the second recombination recognition sequence. between the selectable marker gene and the target gene, and the promoter that regulates the expression of the selectable marker gene and the target gene.
在本发明的一些实施方式中，所述重组酶为Bxb1整合酶、ΦC31整合酶、Cre重组酶或者FLP重组酶。
In some embodiments of the invention, the recombinase is Bxb1 integrase, ΦC31 integrase, Cre recombinase or FLP recombinase.
在本发明的一些实施方式中，所述第一重组识别序列和所述第二重组识别序列独立地选自如下序列中的一种或者多种：所述重组识别序列为LoxP序列、LoxPL3序列、LoxP 2L序列、LoxFas序列、Lox511序列、Lox2272序列、Lox2372序列、Lox5171序列、Loxm2序列、Lox71序列、Lox66序列、FRT序列、Bxb1attP序列、Bxb1attB序列、attP序列和attB序列。In some embodiments of the present invention, the first recombination recognition sequence and the second recombination recognition sequence are independently selected from one or more of the following sequences: the recombination recognition sequence is a LoxP sequence, a LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1attP sequence, Bxb1attB sequence, attP sequence and attB sequence.
在本发明的一些实施方式中，所述选择标记基因选自新霉素抗性基因、胸苷激酶基因、潮霉素磷酸转移酶基因、二氢叶酸还原酶基因、胸苷激酶基因、谷氨酰胺合成酶基因、天冬酰胺合成酶基因、色氨酸合成酶基因、组氨醇脱氢酶基因、氨基糖苷磷酸转移酶基因、色氨酸合成酶基因和荧光蛋白基因中的一种或者多种。In some embodiments of the invention, the selectable marker gene is selected from the group consisting of neomycin resistance gene, thymidine kinase gene, hygromycin phosphotransferase gene, dihydrofolate reductase gene, thymidine kinase gene, glutamine One or more of the amide synthase gene, asparagine synthase gene, tryptophan synthase gene, histidinol dehydrogenase gene, aminoglycoside phosphotransferase gene, tryptophan synthase gene and fluorescent protein gene kind.
在本发明的一些实施方式中，所述启动子为CMV启动子、SV40启动子、RSV启动子、β-globin启动子、UBC启动子、EF1a启动子、泛素启动子、β-actin启动子、PGK1启动子、Rosa26启动子、HSP70启动子、GAPDH启动子、Eif4A1启动子、Egr1启动子、FerH启动子、SM22α启动子或者Endothelin-1启动子。In some embodiments of the invention, the promoter is CMV promoter, SV40 promoter, RSV promoter, β-globin promoter, UBC promoter, EF1a promoter, ubiquitin promoter, β-actin promoter , PGK1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, Eif4A1 promoter, Egr1 promoter, FerH promoter, SM22α promoter or Endothelin-1 promoter.
在本发明的一些实施方式中，所述目标基因编码抗体、重组蛋白、多肽、酶、激素、生长因子和受体中的一种或者多种。In some embodiments of the invention, the target gene encodes one or more of antibodies, recombinant proteins, polypeptides, enzymes, hormones, growth factors and receptors.
本发明的第二方面Second aspect of the invention
本发明提供一种重组载体，所述重组载体包含：The invention provides a recombinant vector, which includes:
a.与SEQ ID No.1所示的核酸片段或者与所述核酸片段保持至少90％一致性的同源片段中所存在的序列片段同源的5’同源臂，a. The 5’ homology arm that is homologous to the sequence fragment present in the nucleic acid fragment shown in SEQ ID No. 1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment,
b.目标基因，b. Target gene,
c.与SEQ ID No.1所示的核酸片段或者与所述核酸片段保持至少90％一致性的同源片段中所存在的序列片段同源的3’同源臂。c. The 3’ homology arm that is homologous to the sequence fragment present in the nucleic acid fragment shown in SEQ ID No. 1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment.
在本发明的一些实施方式中，所述重组载体为慢病毒载体、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体、杆状病毒载体、***瘤病毒载体、***多瘤空泡病毒载体、整合性噬菌体载体、非病毒载体、转座子和/或转座酶、整合酶底物或者质粒。In some embodiments of the invention, the recombinant vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, or a papillomavirus vector. Viral vectors, integrating phage vectors, non-viral vectors, transposons and/or transposases, integrase substrates or plasmids.
本发明的第三方面Third aspect of the invention
本发明提供一种靶向整合细胞，所述靶向整合细胞包含第一方面所述的核酸。The present invention provides a targeted integrating cell, which contains the nucleic acid described in the first aspect.
在本发明的一些实施方式中，所述靶向整合细胞为真核细胞。In some embodiments of the invention, the targeted integration cell is a eukaryotic cell.
在本发明的一些实施方式中，所述真核细胞为哺乳动物细胞。In some embodiments of the invention, the eukaryotic cells are mammalian cells.
在本发明的一些实施方式中，所述哺乳动物细胞包括中国仓鼠卵巢CHO细胞、人胚肾HEK293细胞。In some embodiments of the invention, the mammalian cells include Chinese hamster ovary CHO cells and human embryonic kidney HEK293 cells.
在本发明的一些实施方式中，所述CHO细胞包括CHO宿主细胞、CHO K1宿主细胞、CHO K1SV宿主细胞、DG44宿主细胞、DUKXB-11宿主细胞、CHOK1S宿主细胞或者CHO K1M宿主细胞。In some embodiments of the invention, the CHO cells include CHO host cells, CHO K1 host cells, CHO K1 SV host cells, DG44 host cells, DUKXB-11 host cells, CHOK1S host cells or CHO K1M host cells.
本发明的第四方面Fourth aspect of the invention
本发明提供第三方面所述的靶向整合细胞的制备方法，所述制备方法包括如下步骤：The present invention provides a method for preparing targeted integrated cells according to the third aspect, which method includes the following steps:
将第一方面所述的核酸导入细胞，或者提供包含SEQ ID No.1所示的核酸片段或者与所述核酸片段保持至少90％一致性的同源片段的细胞并将所述外源核酸片段整合至所述SEQ ID No.1所示的核酸片段或者与所述核酸片段保持至少90％一致性的同源片段内，制备靶向整合细胞。Introduce the nucleic acid described in the first aspect into the cell, or provide a cell containing the nucleic acid fragment shown in SEQ ID No. 1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment and use the exogenous nucleic acid fragment Integrate into the nucleic acid fragment shown in SEQ ID No. 1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment to prepare targeted integration cells.
在本发明的一些实施方式中，整合的方式包括但不限于同源重组技术衍生的位点特异性重组技术，依靠针对特异识别位点的整合酶，在基因组和外源DNA间实现基因置换、基因敲出及敲入等基因工程操作，例如可以是重组酶介导的盒式交换，例如可以是CRISPR/Cas9介导的基因靶向整合。In some embodiments of the present invention, the integration method includes but is not limited to site-specific recombination technology derived from homologous recombination technology, which relies on integrase targeting specific recognition sites to achieve gene replacement between the genome and exogenous DNA. Genetic engineering operations such as gene knockout and knock-in can be, for example, recombinase-mediated cassette exchange, or CRISPR/Cas9-mediated gene targeted integration.
本发明的第五方面Fifth aspect of the invention
本发明提供一种生产目标基因表达产物的方法，所述方法包括如下步骤：培养第三方面所述靶向整合细胞，收集目标基因表达产物。The present invention provides a method for producing a target gene expression product, which method includes the following steps: cultivating the targeted integration cells described in the third aspect, and collecting the target gene expression product.
在本发明的一些实施方式中，依照本法的靶向整合细胞具备稳定、高产的特点。In some embodiments of the invention, the targeted integrated cells according to this method are stable and highly productive.
在本发明的一些实施方式中，可以通过实验来鉴定整合位点和/或侧接整合位点的核苷酸序列：在本发明的一些实施方式中，可以通过全基因组筛选方法来鉴定整合位点和/或侧接整合位点的核苷酸序列以分离宿主细胞；在本发明的一些实施方式中，可以在基于转座酶的盒整合事件之后通过全基因组筛选方法来鉴
定整合位点和/或侧接整合位点的核苷酸序列；在本发明的一些实施方式中，可以通过强力(brute force)随机整合筛选来鉴定整合位点和/或侧接整合位点的核苷酸序列；在本发明的一些实施方式中，可以通过常规测序方法(如靶基因座扩增)，之后进行下一代测序和全基因组来确定整合位点和/或侧接整合位点的核苷酸序列；在本发明的一些实施方式中，可以通过常规细胞生物学方法(如荧光原位杂交分析)来确定染色体上整合位点的位置。In some embodiments of the invention, the integration site and/or the nucleotide sequences flanking the integration site can be identified experimentally: In some embodiments of the invention, the integration site can be identified by genome-wide screening methods nucleotide sequences that point and/or flank the integration site to isolate the host cell; in some embodiments of the invention, this can be identified by genome-wide screening methods following a transposase-based cassette integration event. Nucleotide sequences that identify the integration site and/or flank the integration site; in some embodiments of the invention, the integration site and/or flanking integration site can be identified by brute force random integration screening The nucleotide sequence; in some embodiments of the invention, the integration site and/or the flanking integration site can be determined by conventional sequencing methods (such as target locus amplification), followed by next-generation sequencing and whole genome The nucleotide sequence; in some embodiments of the invention, the location of the integration site on the chromosome can be determined by conventional cell biology methods (such as fluorescence in situ hybridization analysis).
具体实施例Specific embodiments
下面将结合实施例对本发明的实施方案进行详细描述。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，优先参考本发明中给出的指引，还可以按照本领域的实验手册或常规条件，还可以按照制造厂商所建议的条件，或者参考本领域已知的实验方法。The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. For experimental methods that do not specify specific conditions in the following examples, priority is given to the guidelines given in the present invention. You can also follow the experimental manuals or conventional conditions in this field. You can also follow the conditions recommended by the manufacturer, or refer to the experimental methods in this field. Known experimental methods.
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。In the following specific examples, the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
下述的具体实施例中的耗材涉及：细胞电转仪专用重悬缓冲液Neon Resuspension Buffer R(ThermoFisher)，电击液E(ThermoFisher)，恢复培养基EX-CELLAdvanced CHO Fed-batch Medium(Sigma-Aldrich)，扩增培养基为EX-CELLAdvanced CHO Fed-batch Medium加200μg/ml hygromycin(ThermoFisher)和1％GlutaMAX(ThermoFisher)，摇瓶培养基为EX-CELLAdvanced CHO Fed-batch Medium加1％GlutaMAX，流加培养基中基础培养基使用100％EX-CELLAdvanced CHO Fed-batch Medium，补料培养基为4％Cell Boost 7a/7b(HyClone)。The consumables in the following specific examples involve: Neon Resuspension Buffer R (ThermoFisher), a special resuspension buffer for cell electroporation apparatus, electroporation solution E (ThermoFisher), recovery medium EX-CELLAdvanced CHO Fed-batch Medium (Sigma-Aldrich) , the amplification medium is EX-CELLAdvanced CHO Fed-batch Medium plus 200 μg/ml hygromycin (ThermoFisher) and 1% GlutaMAX (ThermoFisher), the shake flask medium is EX-CELLAdvanced CHO Fed-batch Medium plus 1% GlutaMAX, fed-batch The basal medium in the culture medium uses 100% EX-CELLAdvanced CHO Fed-batch Medium, and the feed medium is 4% Cell Boost 7a/7b (HyClone).
实施例1Example 1
本实施例的操作流程参见图1，主要包括如下步骤：The operation flow of this embodiment is shown in Figure 1, which mainly includes the following steps:
(1)将绿色荧光蛋白基因(EGFP)作为筛选的标记基因，利用Attp序列作为同源臂，构建一个包含RMCE(图2)的重组质粒，见图3；(1) Use the green fluorescent protein gene (EGFP) as the marker gene for screening, and use the Attp sequence as the homology arm to construct a recombinant plasmid containing RMCE (Figure 2), see Figure 3;
(2)将构建好的质粒扩增、线性化后，用于转染；(2) Amplify and linearize the constructed plasmid and then use it for transfection;
(3)取3×10E6个CHO细胞到50mL离心管，1000rpm室温离心5min，弃上清；(3) Take 3×10E 6 CHO cells into a 50mL centrifuge tube, centrifuge at 1000rpm for 5 minutes at room temperature, and discard the supernatant;
(4)用5mL Ex-Cell Advanced CHO Fed-batch培养基清洗细胞，1000rpm室温离心5min，弃上清；(4) Wash the cells with 5mL of Ex-Cell Advanced CHO Fed-batch medium, centrifuge at 1000rpm for 5 minutes at room temperature, and discard the supernatant;
(5)用细胞电转仪专用重悬缓冲液100μL重悬细胞；(5) Resuspend the cells in 100 μL of special resuspension buffer for cell electroporation;
(6)取线性化后质粒15μg到步骤(5)重悬细胞中，轻柔混匀，吹打50次，避免产生气泡；(6) Take 15 μg of the linearized plasmid and resuspend the cells in step (5), mix gently, and pipet 50 times to avoid bubbles;
(7)打开细胞电转仪，调节参数，电机槽装入电转仪，槽中加入3mL电击液E；(7) Open the cell electroporation instrument, adjust the parameters, install the electroporation instrument into the motor slot, and add 3 mL of electroshock solution E into the tank;
(8)将步骤(6)细胞质粒混悬液吸入电转枪头，装入电击槽中，进行电击；(8) Inhale the cell plasmid suspension in step (6) into the electroporation gun tip, put it into the electroshock tank, and perform electroshock;
(9)将电转后的细胞立即转入加有2mL恢复培养基的6孔板转入37℃5％CO2培养箱过夜培养；(9) Immediately transfer the cells after electroporation into a 6-well plate with 2 mL of recovery medium and culture in a 37°C 5% CO 2 incubator overnight;
(10)电转48h后进行加压筛选，细胞每2-3天传代一次，细胞活率先降低后上升，当细胞活率大于90％时，该细胞为稳定的细胞池；(10) Perform pressurized screening after electroporation for 48 hours. The cells are passaged every 2-3 days. The cell viability first decreases and then increases. When the cell viability rate is greater than 90%, the cells are a stable cell pool;
(11)将稳定细胞池细胞利用流式细胞分选筛选高荧光细胞，获取最高荧光的1％细胞；(11) Use flow cytometric sorting to screen the cells in the stable cell pool for highly fluorescent cells and obtain the 1% cells with the highest fluorescence;
(12)富集后细胞利用精准挑选***荧光排序后进行精准挑选，完成单克隆化；(12) After enrichment, the cells are accurately selected using the precise selection system for fluorescence sorting to complete monoclonalization;
(13)单克隆的荧光细胞，进行扩增培养，形成细胞株，并在摇瓶阶段进行荧光检测，保留高荧光细胞株，记作GBB-01细胞；(13) Monoclonal fluorescent cells are expanded and cultured to form a cell line, and fluorescence detection is performed in the shake flask stage. Highly fluorescent cell lines are retained and recorded as GBB-01 cells;
(14)上述高荧光细胞株进行90天左右的传代稳定性研究，确认荧光值变化较小并且细胞株生长稳定的细胞为稳定高荧光细胞，图4显示GBB-01细胞90天传代生长稳定，表1显示GBB-01细胞荧光表达稳定；(14) The above-mentioned high-fluorescence cell lines were studied for about 90 days of passage stability, and it was confirmed that cells with small changes in fluorescence value and stable cell line growth were stable high-fluorescence cells. Figure 4 shows that GBB-01 cells have stable growth after 90 days of passage. Table 1 shows that the fluorescence expression of GBB-01 cells is stable;
表1
Table 1
Table 1
(15)参照步骤(1)，将目标基因(Fc融合蛋白)片段克隆入质粒中，构建一个包含RMCE(图5)的重组质粒，见图6；(15) Refer to step (1), clone the target gene (Fc fusion protein) fragment into the plasmid, and construct a recombinant plasmid containing RMCE (Figure 5), see Figure 6;
目标基因片段具有SEQ ID No.2所示的序列，SEQ ID No.2：
The target gene fragment has the sequence shown in SEQ ID No. 2, SEQ ID No. 2:
The target gene fragment has the sequence shown in SEQ ID No. 2, SEQ ID No. 2:
(16)选取步骤(14)稳定高荧光细胞GBB-01进行转染、整合验证，将上述步骤(15)重组质粒扩增、线性化后，与Bxb-1整合酶共转染入稳定高荧光细胞GBB-01，转染步骤如前述；(16) Select the stable high-fluorescence cell GBB-01 from step (14) for transfection and integration verification. After amplifying and linearizing the recombinant plasmid from step (15) above, co-transfect it with Bxb-1 integrase into the stable high-fluorescence cell GBB-01. For cells GBB-01, the transfection steps were as described above;
(17)转染2天后细胞池进行加压筛选，2周左右形成稳定细胞池；(17) The cell pool was subjected to pressure screening 2 days after transfection, and a stable cell pool was formed in about 2 weeks;
(18)对细胞池中无荧光细胞比例进行统计，并在细胞活率恢复至90％以上后进行有限稀释法铺板，将细胞池单克隆化；(18) Calculate the proportion of non-fluorescent cells in the cell pool, and after the cell viability returns to more than 90%, perform limiting dilution plating to monoclonal the cell pool;
(19)单克隆细胞中无荧光细胞进行继续扩增(采用扩增培养基)，扩大到摇瓶阶段培养(采用摇瓶培养基)，并通过流加培养(采用流加培养基)进行表达量评估，定点整合的产品单拷贝细胞株(记作产品克隆-1或者GBB-01-H8)与同条件下培养的随机整合的3拷贝细胞株表达量比较结果见图7；(19) The non-fluorescent cells in the monoclonal cells continue to amplify (using amplification medium), expand to the shake flask stage (use shake flask culture medium), and express through fed-batch culture (using fed-batch culture medium) For quantitative evaluation, the comparison results of the expression levels of the single-copy cell line of the product with targeted integration (referred to as product clone-1 or GBB-01-H8) and the randomly integrated 3-copy cell line cultured under the same conditions are shown in Figure 7;
为考察靶向整合细胞产品克隆-1的稳定性，产品克隆-1连续传代90天左右进行稳定性评估，7天的批次培养(只有基础培养基培养)结果产品克隆-1细胞无论从表达量(图8)和比产率(图9)均显示出稳定和高产；In order to examine the stability of the targeted integration cell product Clone-1, the product Clone-1 was continuously passaged for about 90 days for stability evaluation. After 7 days of batch culture (only basal medium culture), the results of the product Clone-1 cells were expressed in terms of expression. Both quantity (Figure 8) and specific yield (Figure 9) showed stable and high yield;
对靶向整合细胞部分质量属性进行了研究，从以下几个方面与随机整合方法[1]得到的细胞株表达产物进行对比：The quality attributes of some targeted integrated cells were studied, and compared with the cell line expression products obtained by the random integration method [1] from the following aspects:
结果显示质量属性接近，没有显著差异，见图10糖谱对比结果和图11电荷异质性对比结果；The results show that the quality attributes are close and there is no significant difference. See Figure 10 for sugar spectrum comparison results and Figure 11 for charge heterogeneity comparison results;
对荧光、目标基因均高表达细胞株GBB-01-H8进行二代全基因组测序，获取整合位点在CHO上的注释信息如下：NC_048596:34375010位于基因间区。Second-generation whole-genome sequencing was performed on GBB-01-H8, a cell line with high expression of both fluorescence and target genes, and the annotation information of the integration site on CHO was obtained as follows: NC_048596:34375010 is located in the intergenic region.
整合目标基因后的NC_048596序列片段如SEQ ID No.3所示，SEQ ID No.3：
The NC_048596 sequence fragment after integrating the target gene is shown in SEQ ID No.3, SEQ ID No.3:
The NC_048596 sequence fragment after integrating the target gene is shown in SEQ ID No.3, SEQ ID No.3:
用上述断点上游引物F(SEQ ID No.4):tgcagccacactaagatgg与产品所用质粒上产品基因下游引物R(SEQ ID No.5)：ccagagacaggctcaaggac扩增该产品细胞株基因组，得到符合理论大小(2876bp)的片段，如图12(GBB-01-H8泳道对应条带)所示。Use the above breakpoint upstream primer F (SEQ ID No. 4): tgcagccacactaagatgg and the product gene downstream primer R (SEQ ID No. 5): ccagagacaggctcaaggac on the plasmid used in the product to amplify the genome of the product cell line, and obtain a protein that conforms to the theoretical size (2876bp) The fragment is shown in Figure 12 (corresponding band of GBB-01-H8 lane).
将扩增出来的条带切胶回收，纯化，并设计多对引物进行DNA测序，测序结果拼接后序列如下所示，和理论序列一致(序列SEQ ID No.3)，从而证实目标基因正确整合入目标位点。The amplified bands were cut and purified, and multiple pairs of primers were designed for DNA sequencing. The sequence after splicing of the sequencing results is as follows, which is consistent with the theoretical sequence (sequence SEQ ID No. 3), thus confirming that the target gene is correctly integrated. into the target site.
参考文献：references:
[1]Takeshi Omasa et al,.Cell engineering and cultivation of chinese hamster ovary(CHO)cells.Curr Pharm Biotechnol.2010 Apr；11(3):233-40.[1]Takeshi Omasa et al,.Cell engineering and cultivation of chinese hamster ovary(CHO)cells.Curr Pharm Biotechnol.2010 Apr;11(3):233-40.
实施例2Example 2
(1)构建Cas9编辑质粒：(1) Construct Cas9 editing plasmid:
设计可以靶向SEQ ID No.1所示的核酸片段第1678-1744个碱基区间(ttttaccacaactgatagtgtaagaaaacaaactgaagtagatagtcacccacaccagagacaaag)具有较高剪切效率的sgRNA(SEQ ID No.6：gatagtcacccacaccagaggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc；GC＝55％，无二级)，双链合并退火。反应体系为正反向oligo各5μL，5μL 10×NEB buffer 2，用水补至50μL。体系于95℃(必须用温度计量取)孵育5min，之后自然降温至25℃。得到的产物质粒和PvuI酶切过的pDonor CRISPR/Cas9载体连接。连接产物转化于DH5α感受态中，涂于氨苄抗性的LB固体平板，次日挑取单克隆，并抽取质粒。最后通过一代测序验证得到正确的Cas9编码质粒。Design an sgRNA that can target the 1678-1744th base interval of the nucleic acid fragment shown in SEQ ID No. 1 (ttttaccacaactgatagtgtaagaaaacaaactgaagtagatagtcacccacaccagagacaaag) with higher shearing efficiency (SEQ ID No. 6: gatagtcacccacaccagaggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttga aaaagtggcaccgagtcggtgc; GC=55%, no secondary ), double-stranded merger annealing. The reaction system is 5 μL of forward and reverse oligo, 5 μL of 10×NEB buffer 2, and make up to 50 μL with water. The system was incubated at 95°C (temperature measurement must be used) for 5 minutes, and then naturally cooled to 25°C. The obtained product plasmid was ligated with the pDonor CRISPR/Cas9 vector digested by PvuI. The ligation product was transformed into DH5α competent cells and spread on an ampicillin-resistant LB solid plate. The next day, single clones were picked and the plasmid was extracted. Finally, the correct Cas9 encoding plasmid was verified through first-generation sequencing.
(2)构建供体质粒：(2) Construct donor plasmid:
供体质粒由5’同源臂、anti-PD1单克隆抗体编码基因，潮霉素编码基因和3’同源臂串联于pUC19质粒上构成。The donor plasmid consists of a 5' homology arm, an anti-PD1 monoclonal antibody coding gene, a hygromycin coding gene and a 3' homology arm connected in series on the pUC19 plasmid.
(3)电穿孔转染Cas9编码质粒和anti-PD1供体质粒：(3) Electroporation and transfection of Cas9 encoding plasmid and anti-PD1 donor plasmid:
使用悬浮型的中国仓鼠卵巢癌细胞，即CHOK1细胞株。转染前一天将细胞密度调整为5×105个/mL。第二天使用电穿孔的方法进行转染，条件为电压1575V，脉冲时程10ms，脉冲个数3个，转染使用细胞培养的6孔板，3×106个细胞，培养基体积为2mL，所需质粒为15μg。转染完成后放置于37℃、5％CO2的培养箱中培养。其中，加入Cas9编码质粒和anti-PD1供体质粒进行共转染可以得到定点整合细胞池，仅加入anti-PD1供体质粒转染可以得到表达anti-PD1的随机整合细胞池。Suspended Chinese hamster ovary cancer cells, CHOK1 cell line, were used. The day before transfection, the cell density was adjusted to 5 × 10 5 cells/mL. The electroporation method was used for transfection the next day. The conditions were voltage 1575V, pulse duration 10ms, and pulse number 3. Transfection used a 6-well cell culture plate, 3×10 6 cells, and a culture medium volume of 2 mL. , the required plasmid is 15μg. After the transfection is completed, place it in an incubator at 37°C and 5% CO2 for culture. Among them, adding Cas9 encoding plasmid and anti-PD1 donor plasmid for co-transfection can obtain a site-directed integrated cell pool, and only adding anti-PD1 donor plasmid for transfection can obtain a randomly integrated cell pool expressing anti-PD1.
(4)加压筛选并富集定点整合的细胞池。(4) Pressure screening and enrichment of site-specific integrated cell pools.
(5)5’/3’junction PCR初步鉴定定点整合细胞池，对扩增得到的目标片段测序，确定得到了定点整合细胞池后，收集上清。(5) 5’/3’junction PCR is used to initially identify the site-specific integrated cell pool, and the amplified target fragment is sequenced. After confirming that the site-directed integrated cell pool is obtained, the supernatant is collected.
(6)Western blot检测定点整合细胞池anti-PD1抗体的表达，确定得到了定点整合细胞池。随机整合细胞池在加药筛选3周后通过Western blot基本检测不到anti-PD1抗体的表达，而定点整合细胞池的anti-PD1抗体表达量较高。(6) Western blot detects the expression of anti-PD1 antibody in the site-directed integrated cell pool and confirms that the site-directed integrated cell pool is obtained. The expression of anti-PD1 antibody in the randomly integrated cell pool was basically undetectable by Western blot 3 weeks after drug addition and screening, while the anti-PD1 antibody expression in the fixed-point integrated cell pool was higher.
(7)有限稀释法获得单克隆细胞株：将之前的定点整合细胞池通过有限稀释法获得单克隆细胞株。即将细胞池铺于96孔板，细胞密度为0.8个/孔。当天查板，确定单克隆。细胞生长约5周时，将单克隆细胞吸取1×106个并提取基因组DNA，再次进行5’/3’junction PCR检测，方法同步骤(5)。将Junction PCR检测得到阳性细胞株进一步进行Western blot检测，判断anti-PD1抗体是否正确表达。(7) Obtain monoclonal cell lines by limiting dilution method: Use the previous site-specific integrated cell pool to obtain monoclonal cell lines by limiting dilution method. That is, the cell pool was spread on a 96-well plate at a cell density of 0.8 cells/well. Check the board on the same day to determine the single clone. When the cells grow for about 5 weeks, aspirate 1×10 6 monoclonal cells and extract genomic DNA, and perform 5'/3'junction PCR detection again, using the same method as step (5). The positive cell lines detected by Junction PCR were further tested by Western blot to determine whether the anti-PD1 antibody was correctly expressed.
(8)定点整合细胞株anti-PD1抗体表达量的评估：靶向整合细胞株的anti-PD1抗体表达量约为随机
整合细胞株表达量的2.3倍。(8) Evaluation of anti-PD1 antibody expression in site-specific integration cell lines: The expression of anti-PD1 antibodies in targeted integration cell lines is approximately random The expression level of the integrated cell line was 2.3 times.
实施例3Example 3
表2
Table 2
Table 2
依实施例1步骤构建靶向整合细胞，以单拷贝随机整合的细胞株为对照，于基础培养基中，6孔板批次培养的表达量见表2，其中单拷贝随机整合的细胞株anti-PD1抗体在6孔板批次培养表达量为285mg/L，而靶向整合细胞株的表达量为其2倍以上水平。Targeted integration cells were constructed according to the steps of Example 1. The cell lines with single copy random integration were used as controls. In the basic medium, the expression levels of 6-well plate batch culture were shown in Table 2. The cell lines with single copy random integration anti -The expression level of PD1 antibody in 6-well plate batch culture is 285 mg/L, while the expression level of the targeted integration cell line is more than 2 times that level.
以上所述实施方式和实施例的各技术特征可以进行任意合适方式的组合，为使描述简洁，未对上述实施方式和实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为在本说明书记载的范围中。The technical features of the above-described embodiments and examples can be combined in any suitable manner. To simplify the description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as these There is no contradiction in the combination of technical features, and they should be considered to be within the scope of this specification.
以上所述实施例仅表达了本发明的几种实施方式，便于具体和详细地理解本发明的技术方案，但并不能因此而理解为对发明专利保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，得到的等价形式同样落于本发明的保护范围。还应当理解，本领域技术人员在本发明提供的技术方案的基础上，通过合乎逻辑的分析、推理或者有限的试验得到的技术方案，均在本发明所附权利要求的保护范围内。因此，本发明专利的保护范围应以所附权利要求的内容为准，说明书及附图可以用于解释权利要求的内容。
The above-mentioned embodiments only express several implementation modes of the present invention to facilitate a specific and detailed understanding of the technical solutions of the present invention, but they should not be construed as limiting the scope of protection of the invention patent. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited testing based on the technical solutions provided by the present invention are within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the contents of the appended claims, and the description and drawings may be used to interpret the contents of the claims.
Claims (13)
- 核酸，其特征在于，所述核酸包含SEQ ID No.1所示的核酸片段，所述核酸片段内整合有外源核酸片段。Nucleic acid, characterized in that the nucleic acid includes the nucleic acid fragment shown in SEQ ID No. 1, and the exogenous nucleic acid fragment is integrated into the nucleic acid fragment.
- 根据权利要求1所述的核酸，其特征在于，所述外源核酸片段的整合位点对应所述核酸片段的第100-3000个碱基区间内的任意位点；The nucleic acid according to claim 1, wherein the integration site of the exogenous nucleic acid fragment corresponds to any position within the 100-3000th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第150-2820个碱基区间内的任意位点；Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 150-2820th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第324-2688个碱基区间内的任意位点；Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 324-2688th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第480-2460个碱基区间内的任意位点；Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 480-2460th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第700-2200个碱基区间内的任意位点；Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 700-2200th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第976-1900个碱基区间内的任意位点；Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 976-1900th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第1260-1879个碱基区间内的任意位点；Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1260-1879th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第1561-1800个碱基区间内的任意位点；Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1561-1800th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第1678-1744个碱基区间内的任意位点；Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1678-1744th base interval of the nucleic acid fragment;可选地，所述外源核酸片段的整合位点对应所述核酸片段的第1701-1720个碱基区间内的任意位点。Optionally, the integration site of the exogenous nucleic acid fragment corresponds to any position within the 1701-1720th base interval of the nucleic acid fragment.
- 根据权利要求1或者2所述的核酸，其特征在于，所述外源核酸片段包含：重组酶识别的第一重组识别序列和第二重组识别序列，位于所述第一重组识别序列和所述第二重组识别序列之间的选择标记基因和目标基因，以及调控所述选择标记基因和所述目标基因表达的启动子。The nucleic acid according to claim 1 or 2, characterized in that the exogenous nucleic acid fragment includes: a first recombination recognition sequence and a second recombination recognition sequence recognized by a recombinase, located between the first recombination recognition sequence and the The second recombination recognition sequence is between the selectable marker gene and the target gene, and the promoter that regulates the expression of the selectable marker gene and the target gene.
- 根据权利要求3所述的核酸，其特征在于，所述重组酶为Bxb1整合酶、ΦC31整合酶、Cre重组酶或者FLP重组酶；或/和，The nucleic acid according to claim 3, characterized in that the recombinase is Bxb1 integrase, ΦC31 integrase, Cre recombinase or FLP recombinase; or/and,所述第一重组识别序列和所述第二重组识别序列独立地选自如下序列中的一种或者多种：LoxP序列、LoxPL3序列、LoxP 2L序列、LoxFas序列、Lox511序列、Lox2272序列、Lox2372序列、Lox5171序列、Loxm2序列、Lox71序列、Lox66序列、FRT序列、Bxb1 attP序列、Bxb1 attB序列、attP序列和attB序列。The first recombination recognition sequence and the second recombination recognition sequence are independently selected from one or more of the following sequences: LoxP sequence, LoxPL3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence , Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, attP sequence and attB sequence.
- 根据权利要求3所述的核酸，其特征在于，所述选择标记基因选自新霉素抗性基因、胸苷激酶基因、潮霉素磷酸转移酶基因、二氢叶酸还原酶基因、胸苷激酶基因、谷氨酰胺合成酶基因、天冬酰胺合成酶基因、色氨酸合成酶基因、组氨醇脱氢酶基因、氨基糖苷磷酸转移酶基因、色氨酸合成酶基因和荧光蛋白基因中的一种或者多种。The nucleic acid according to claim 3, characterized in that the selectable marker gene is selected from the group consisting of neomycin resistance gene, thymidine kinase gene, hygromycin phosphotransferase gene, dihydrofolate reductase gene, thymidine kinase gene, glutamine synthetase gene, asparagine synthase gene, tryptophan synthase gene, histidinol dehydrogenase gene, aminoglycoside phosphotransferase gene, tryptophan synthase gene and fluorescent protein gene One or more.
- 根据权利要求3所述的核酸，其特征在于，所述启动子为CMV启动子、SV40启动子、RSV启动子、β-globin启动子、UBC启动子、EF1a启动子、泛素启动子、β-actin启动子、PGK1启动子、Rosa26启动子、HSP70启动子、GAPDH启动子、Eif4A1启动子、Egr1启动子、FerH启动子、SM22α启动子或者Endothelin-1启动子。The nucleic acid according to claim 3, characterized in that the promoter is CMV promoter, SV40 promoter, RSV promoter, β-globin promoter, UBC promoter, EF1a promoter, ubiquitin promoter, β -actin promoter, PGK1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, Eif4A1 promoter, Egr1 promoter, FerH promoter, SM22α promoter or Endothelin-1 promoter.
- 根据权利要求3所述的核酸，其特征在于，所述目标基因编码抗体、重组蛋白、多肽、酶、激素、生长因子和受体中的一种或者多种。The nucleic acid according to claim 3, wherein the target gene encodes one or more of antibodies, recombinant proteins, polypeptides, enzymes, hormones, growth factors and receptors.
- 重组载体，其特征在于，所述载体包含：Recombinant vector, characterized in that the vector contains:a.与SEQ ID No.1所示的核酸片段所存在的序列片段同源的5’同源臂，a. The 5’ homology arm that is homologous to the sequence fragment present in the nucleic acid fragment shown in SEQ ID No. 1,b.目标基因，b. Target gene,c.与SEQ ID No.1所示的核酸片段所存在的序列片段同源的3’同源臂。c. The 3’ homology arm that is homologous to the sequence fragment present in the nucleic acid fragment shown in SEQ ID No. 1.
- 根据权利要求8所述的重组载体，其特征在于，所述重组载体为慢病毒载体、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体、杆状病毒载体、***瘤病毒载体、***多瘤空泡病毒载体、整合性噬菌体载体、非病毒载体、转座子和/或转座酶、整合酶底物或者质粒。The recombinant vector according to claim 8, characterized in that the recombinant vector is a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, Papilloma vacuolating viral vectors, integrating phage vectors, non-viral vectors, transposons and/or transposases, integrase substrates or plasmids.
- 靶向整合细胞，其特征在于，所述靶向整合细胞包含权利要求1至7任一项所述的核酸。Targeted integration cell, characterized in that the targeted integration cell contains the nucleic acid according to any one of claims 1 to 7.
- 根据权利要求10所述的靶向整合细胞，其特征在于，所述靶向整合细胞为真核细胞；The targeted integration cell according to claim 10, characterized in that the targeted integration cell is a eukaryotic cell;可选地，所述真核细胞为哺乳动物细胞；Optionally, the eukaryotic cells are mammalian cells;可选地，所述哺乳动物细胞包括中国仓鼠卵巢CHO细胞、人胚肾HEK293细胞。Optionally, the mammalian cells include Chinese hamster ovary CHO cells and human embryonic kidney HEK293 cells.
- 权利要求10或者11所述的靶向整合细胞的制备方法，其特征在于，所述制备方法包括如下步骤：The preparation method of targeted integrated cells according to claim 10 or 11, characterized in that the preparation method includes the following steps:将权利要求1至7任一项所述的核酸导入细胞，或者提供包含SEQ ID No.1所示的核酸片段的细胞并将所述外源核酸片段整合至所述核酸片段，制备靶向整合细胞。Introduce the nucleic acid described in any one of claims 1 to 7 into a cell, or provide a cell containing the nucleic acid fragment shown in SEQ ID No. 1 and integrate the exogenous nucleic acid fragment into the nucleic acid fragment to prepare targeted integration cell.
- 生产目标基因表达产物的方法，其特征在于，所述方法包括如下步骤：培养权利要求10或者11所 述的靶向整合细胞，收集所述外源核酸片段中目标基因的表达产物。 A method for producing target gene expression products, characterized in that the method includes the following steps: cultivating the gene expression product according to claim 10 or 11 The targeted integration cell described above is used to collect the expression product of the target gene in the exogenous nucleic acid fragment.
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