Mammalian cells that highly express EPO protein

Abstract

The present invention generally relates to the field of biotechnology, and more specifically, to a mammalian cell that highly expresses EPO protein. The cell has a gene expression cassette containing one or more copies of the epo gene inserted into the third intron of the Mast2 gene, and can stably express the EPO protein. The gene insertion site provided by the present invention has the advantages of high expression and stable passage.

Description

Technical Field

[0001] The present invention generally relates to the field of biotechnology, and in particular, to a mammalian cell that highly expresses EPO protein. Background Art

[0002] Erythropoietin (EPO) is a highly sialylated glycoprotein hormone mainly secreted by the kidneys. It acts on erythroid hematopoietic progenitor cells in the bone marrow, promoting their proliferation and differentiation, and increasing the body's hemoglobin concentration. Recombinant EPO protein is expressed by Chinese hamster ovary (CHO) cells. Since it was developed and marketed by Amgen Biotech in the United States in 1989, it has quickly become the most effective and highest-selling bioengineered drug in the world.

[0003] CHO cells are a widely used mammalian expression system in the biopharmaceutical field. The recombinant proteins produced by them have post-translational modifications similar to those of humanized cells. About 70% of recombinant protein drugs are produced by CHO cells. The post-translational glycosylation modification of EPO recombinant protein is very critical and directly affects its half-life and immunogenicity in the human body.

[0004] The traditional method of constructing a cell line expressing an exogenous protein is to randomly insert it into the cell genome. Due to the randomness of the integration site and the number of gene copies, there is a risk of copy number loss during long-term industrial production. Since there are huge differences in expression levels between randomly integrated monoclonal cell lines, a lot of manpower, material resources, and time are required to screen monoclonal cell lines. Summary of the invention

[0005] The present invention covers the following technical solutions:

[0006] The first object of the present invention is to provide a mammalian cell. Mast2 One or more insertions into the third intron of the gene epo The gene expression cassette of the gene copy can stably express EPO protein.

[0007] The gene insertion site provided by the present invention has the advantages of high expression level and stable transmission.

[0008] In some embodiments, the copy number is 1, 2, 3 or more.

[0009] In some embodiments, the gene expression cassette further comprises one or more elements selected from the group consisting of:

[0010] i) Promoter;

[0011] ii) signal peptide coding sequence;

[0012] iii) leader coding sequence;

[0013] iv) terminator;

[0014] v) marker genes;

[0015] vi) Polyadenylation sequence.

[0016] The marker gene in the present invention is broad in scope, and includes genes used for screening (e.g., antibiotic resistance genes) and marker genes, etc. Commonly used marker genes are fragments of nucleic acids, etc., for generating fluorescent proteins. Fluorescent proteins can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein.

[0017] Green fluorescent protein can use common GFP, or a modified GFP gene, such as enhanced GFP gene EGFP, etc.; blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein can be selected from EYFP, Ypct, PhiYFP, etc.; orange fluorescent protein can be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate, etc.

[0018] In some embodiments, the epo The amino acid sequence of the gene is shown in SEQ ID NO:1.

[0019] In some embodiments, the mammalian cell is a cell line.

[0020] In some embodiments, the mammalian cell is a CHO-K1 cell line.

[0021] In some embodiments, the insertion position of the gene expression cassette is located by the upstream LP-homologous arm shown in SEQ ID NO:2 and the downstream LP-homologous arm shown in SEQ ID NO:3.

[0022] The second object of the present invention is to provide a nucleic acid construct comprising the upstream LP-homologous arm shown in SEQ ID NO: 2, epo The gene expression cassette of the gene and the downstream LP-homologous arm shown in SEQ ID NO:3; wherein the epo The gene expression cassette of the gene is as defined above for the gene expression cassette.

[0023] In some embodiments, the nucleic acid construct further comprises attP and attB sites, and a gene sequence of BxB1 recombinase.

[0024] Preferably, attP has the nucleotide sequence shown in SEQ ID NO:5.

[0025] Preferably, attB has the nucleotide sequence shown in SEQ ID NO:6.

[0026] The third object of the present invention is to provide a vector comprising the nucleic acid construct as described above.

[0027] The term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40), and insect baculoviruses.

[0028] The fourth object of the present invention is to provide a gene editing combination product, comprising:

[0029] a) a vector as described above; and

[0030] b) Nucleic acid cleavage system, its target recognition Mast2 The specific sequence of the third intron of the gene; and enables the nick to be inserted by the nucleic acid construct in the form of homologous recombination.

[0031] In some embodiments, the nucleic acid cleavage system is a CRISPR / Cas9 system.

[0032] In some embodiments, the sgRNA sequence of the nucleic acid cleavage system is shown in SEQ ID NO:4.

[0033] A fifth object of the present invention is to provide a method for preparing EPO protein, which comprises:

[0034] i) culturing the mammalian cells as described above to express EPO protein;

[0035] ii) Disrupt the cells and isolate the EPO protein from them.

[0036] For step ii), if necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0037] The cells provided by the present invention can be used to stably and highly express long-acting recombinant EPO protein.

[0038] The construction method preferably uses site-specific integration technology to precisely insert heterologous DNA into pre-verified loci, allowing mammalian cell lines to stably express exogenous genes. The specific integration platform allows the controllable insertion of DNA into stable genomic sites and has a wide range of applications in synthetic biology, recombinant protein production and biomanufacturing. epo The precise integration of genes into stable and highly expressed sites within the cell genome can simplify the screening process of monoclonal cell lines and improve screening efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 :Schematic diagram of knock-in donor LP donor.

[0041] Figure 2 :Flow cytometry analysis results of CHO-K1 LP-m2 steady-state cell monoclonal cell line;

[0042] The horizontal axis is the mCherry red fluorescence detected by the PE-A channel. The light color represents the fluorescence peak of the CHO-K1 control cells, and the dark color represents the fluorescence peak of the LP-m2 steady-state cell monoclonal cell line.

[0043] Figure 3 : Schematic diagram of payload integration with landing pad at specific genome sites.

[0044] Figure 4 :Schematic diagram of lentiviral vector payload and BxB1.

[0045] Figure 5:The results of EPO expression detection in CHO-K1 LP-m2 site-specific integration and monoclonal cell lines screened by traditional methods.

[0046] Figure 6 :Stability test results of EPO expression in CHO-K1 LP-m2 site-specific integration and monoclonal cell lines screened by traditional methods;

[0047] After 10 consecutive passages and 6 days of culture, EPO expression was detected by Western blot. Lanes 1-11 are monoclonal cell lines screened by traditional methods; lanes 12-14 are EPO cell lines with a single gene copy obtained by site-directed integration of CHO-K1 LP-m2.

[0048] Figure 7 :EPO expression detection results of different gene copy numbers in CHO-K1 LP-m2 cells;

[0049] The EPO expression was detected by Western blot; Lane 1 is a cell line expressing EPO with a single gene copy; Lane 2 is a cell line expressing EPO with three gene copies.

[0050] Figure 8 :EPO expression test results of CHO-K1 LP-m2 site-specific integration and other site-specific integration cell lines;

[0051] Western blot was used to detect EPO expression; Lanes 1-2 are EPO expression cell lines obtained by site-directed integration of CHO-K1 LP-m2 with a single gene copy; Lanes 3-5 are CHO-K1 Clcc1 An EPO expressing cell line with a single gene copy obtained by site-specific integration of exon 11 of the gene. DETAILED DESCRIPTION

[0052] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0053] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0054] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0055] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present invention, the technical solution undoubtedly includes technical solutions connected by "logical and", and undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0056] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0057] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0058] As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0059] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0060] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0061] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0062] In the present invention, "preferably", "better", "more preferably", and "suitably" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent of each other.

[0063] All documents mentioned in the present invention are cited as references in the present invention, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present invention, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features can also be incorporated into the present invention as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in the present invention, the present invention shall prevail or be modified adaptively according to the description of the present invention.

[0064] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0065] The present application relates to a mammalian cell, which Mast2 One or more insertions into the third intron of the gene epo The gene expression cassette of the gene copy can stably express EPO protein.

[0066] The gene insertion site provided by the present invention has the advantages of high expression level and stable transmission.

[0067] In some embodiments, the copy number is 1, 2, 3 or more.

[0068] In some embodiments, the gene expression cassette further comprises one or more elements selected from the group consisting of:

[0069] i) Promoter;

[0070] ii) signal peptide coding sequence;

[0071] iii) leader coding sequence;

[0072] iv) terminator;

[0073] v) marker genes;

[0074] vi) Polyadenylation sequence.

[0075] The marker gene in the present invention is broad in scope, and includes genes used for screening (e.g., antibiotic resistance genes) and marker genes, etc. Commonly used marker genes are fragments of nucleic acids, etc., for generating fluorescent proteins. Fluorescent proteins can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, or red fluorescent protein.

[0076] Green fluorescent protein can use common GFP, or a modified GFP gene, such as enhanced GFP gene EGFP, etc.; blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein can be selected from EYFP, Ypct, PhiYFP, etc.; orange fluorescent protein can be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate, etc.

[0077] In some embodiments, the amino acid sequence of the epo gene is as shown in SEQ ID NO:1.

[0078] In some embodiments, the mammalian cell is a cell line.

[0079] In some embodiments, the mammalian cell is a CHO-K1 cell line.

[0080] In some embodiments, the insertion position of the gene expression cassette is located by the upstream LP-homologous arm shown in SEQ ID NO:2 and the downstream LP-homologous arm shown in SEQ ID NO:3.

[0081] The second object of the present invention is to provide a nucleic acid construct, which comprises an upstream LP-homologous arm shown in SEQ ID NO:2, a gene expression cassette of the epo gene, and a downstream LP-homologous arm shown in SEQ ID NO:3, which are connected in sequence; wherein the gene expression cassette of the epo gene is as defined in the gene expression cassette above.

[0082] In some embodiments, the nucleic acid construct further comprises attP and attB sites, and a gene sequence of BxB1 recombinase.

[0083] Preferably, attP has the nucleotide sequence shown in SEQ ID NO:5.

[0084] Preferably, attB has the nucleotide sequence shown in SEQ ID NO:6.

[0085] The third object of the present invention is to provide a vector comprising the nucleic acid construct as described above.

[0086] The term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40), and insect baculoviruses.

[0087] The fourth object of the present invention is to provide a gene editing combination product, comprising:

[0088] a) a vector as described above; and

[0089] b) a nucleic acid cleavage system that targets and recognizes a specific sequence in the third intron of the Mast2 gene and enables the nucleic acid construct to be inserted into the nick in the form of homologous recombination.

[0090] In some embodiments, the nucleic acid cleavage system is a CRISPR / Cas9 system.

[0091] In some embodiments, the sgRNA sequence of the nucleic acid cleavage system is shown in SEQ ID NO:4.

[0092] A fifth object of the present invention is to provide a method for preparing EPO protein, which comprises:

[0093] i) culturing the mammalian cells as described above to express EPO protein;

[0094] ii) Disrupt the cells and isolate the EPO protein from them.

[0095] For step ii), if necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0096] The cells provided by the present invention can be used to stably and highly express long-acting recombinant EPO protein.

[0097] The construction method preferably uses site-specific integration technology to precisely insert heterologous DNA into pre-verified loci, so that mammalian cell lines can stably express exogenous genes. The specific integration platform allows the controllable insertion of DNA into stable genomic sites and has a wide range of applications in synthetic biology, recombinant protein production and biomanufacturing. Using site-specific integration technology to precisely integrate the codon-optimized long-acting recombinant epo gene into a stable and highly expressed site in the cell genome can simplify the screening process of monoclonal cell lines and improve screening efficiency.

[0098] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0099] Example 1 - Site-directed integration of CHO-K1 LP-m2 cells

[0100] To achieve stable expression of foreign genes in mammalian cell lines, it is necessary to precisely insert foreign DNA into well-characterized genomic sites. A genomic site that supports long-term stable expression of transgenes was selected to construct a CHO-K1 LP-m2 monoclonal cell line containing a "landing pad" (LP) recombination site.

[0101] The specific steps are as follows:

[0102] LP-m2 steady-state cells were constructed using the CRISPR / Cas9 system. The genomic DNA at the selected site was cut using the Cas9 protein, and the LP donor was inserted into the cut genome through homologous recombination to build a site-specific integration platform for exogenous genes. The LP-m2 gRNA / Cas9 plasmid was constructed using pSpCas9(BB)-2A-Puro (PX459) V2.0 as a vector, and the gRNA was introduced in front of the gRNA-scaffold.

[0103] LP donor is used as a gene knock-in donor for CRISPR / Cas9 system. Both ends of the fragment have homology arms of the genome. Based on the homology sequence, the fragment can be inserted into the target location of the genome. The landing pad of LP donor is composed of CMV promoter, BxB1 recombinase recognition site attP sequence, reporter gene mCherry and the SV40 polyA signal sequence. mCherry Transcription is controlled by the strong CMV promoter, and red fluorescence can be used to screen LP-m2 steady-state cell clones and characterize the expression of exogenous genes at selected genomic loci.

[0104] The LP-m2 gRNA / Cas9 plasmid and the purified LP donor fragment were co-transfected into CHO-K1 cells at a mass ratio of 1:1. The co-transfected CHO-K1 LP-m2 cells were flow sorted based on red fluorescence to obtain mCherry-positive cells. Monoclonal clones were isolated by limiting dilution and monoclonal cell lines that stably expressed red fluorescence were selected.

[0105] Example 2 - Construction of EPO high-expressing cell line using site-directed integration technology

[0106] Based on CHO-K1 LP-m2 steady-state cell platform, epo Multiple copies of the gene can be inserted into a selected site through BxB1 recombinase, achieving controllable integration.

[0107] The payload is designed as follows:

[0108] The attB site is placed before the resistance gene, replacing its promoter. When the payload plasmid is co-transfected with the BxB1 recombinase plasmid into LP-m2 steady-state cells, the BxB1 enzyme cuts the attP and attB sites, allowing the linearized payload to be inserted into the intended location. The resistance gene in the payload is expressed using the CMV promoter of the landing pad, showing drug resistance, and the required polyclonal cells can be screened by drug killing. The expression of EPO depends on its own promoter. After integration, the PolyA sequence of the payload causes the downstream mCherry to no longer be expressed, and the red fluorescence of the cells disappears. This can be used as a judgment epo One of the bases for successful gene integration.

[0109] In order to further improve the efficiency of transfection and integration, lentivirus was used to deliver the payload fragment and BxB1 gene. The design is as follows:

[0110] The pCDH vector contains CMV and EF1α dual promoters. The BxB1 gene is inserted into the EF1α promoter, and the CMV promoter of the vector is replaced by the payload fragment. Because the payload fragment contains the transcription termination signal PolyA, it is inserted into the pCDH vector in reverse during construction to avoid RNA error termination during lentiviral packaging. epo Polyclonal cells with successful gene integration.

[0111] Example 3 - Isolation and identification of EPO high-expressing cell lines

[0112] Transfection with PEI or Lipo6000 epo The gene (1 or 3 copies) is integrated into the cell genome. After culturing the transfected cells for 48 hours, 1.5 mg / mL G418 is used for drug killing screening, and the culture medium is replaced every 2-3 days. After culturing for about 7 days, 95% of the cells in the negative control group fell off and died under an inverted fluorescence microscope. After continuing to culture with a culture medium containing 1.5 mg / mL G418 for 3 days, a cell monoclonal group is formed. The transfected CHO-K1 LP-m2 cells are flow sorted based on red fluorescence. Red is a false positive. The positive cells expressing EPO are replaced by mCherry Genes are displayed as colorless. Colorless monoclonal cells were sorted into 96-well plates and inoculated into 48-well plates after confluence. After 6 days of culture, the culture medium was aspirated and the expression level was detected by Western Blotting method. Figure 5-7 shown.

[0113] In order to further verify the effect of the present invention, the inventors designed a control example. The control example comes from Gaidukov L, Wroblewska L, Teague B, et al. A multi-landing pad DNA integration platform for mammalian cell engineering[J]. Nucleic acids research, 2018, 46(8): 4072-4086. Clcc1 The 11th exon of the gene was site-specifically integrated, but the relevant expression system was set up using the example of this application. Figure 8 As shown, compared with the control example, the insertion site of the present invention was significantly improved.

[0114] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A CHO-K1 cell line, characterized in that: Its Mast2 The third intron of the gene contains one or more epo A gene expression cassette containing a gene copy and capable of stably expressing EPO protein, epo The amino acid sequence of the gene is shown in SEQ ID NO:

1.

2. The CHO-K1 cell line according to claim 1, characterized in that The gene expression cassette further comprises one or more elements selected from the group consisting of: i) Promoter; ii) signal peptide coding sequence; iii) leader coding sequence; iv) terminator; v) marker genes; vi) Polyadenylation sequence.

3. The CHO-K1 cell line according to claim 1 or 2, characterized in that: The copy number is 1, 2, 3 or more.

4. A nucleic acid construct, characterized in that comprising the upstream LP-homologous arm shown in SEQ ID NO: 2 connected in sequence, epo The gene expression cassette of the gene and the downstream LP-homologous arm shown in SEQ ID NO:3; wherein the epo The gene expression cassette of a gene is as defined in any one of claims 1 to 3.

5. The nucleic acid construct according to claim 4, characterized in that It also contains attP and attB sites, and the gene sequence for the BxB1 recombinase.

6. A carrier, characterized in that Comprising the nucleic acid construct according to claim 4 or 5.

7. A gene editing combination product, characterized in that: Include: a) the vector according to claim 6; and b) Nucleic acid cleavage system, its target recognition Mast2 The specific sequence of the third intron of the gene; and enables the nick to be inserted by the nucleic acid construct in the form of homologous recombination.

8. The gene editing combination product according to claim 7, characterized in that: The nucleic acid cleavage system is a CRISPR / Cas9 system; its sgRNA sequence is shown in SEQ ID NO:

4.

9. A method for preparing EPO protein, characterized in that: include: i) culturing the CHO-K1 cell line according to any one of claims 1 to 3 to express EPO protein; ii) Disrupt the cells and isolate the EPO protein from them.

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