Screening method of site-specific integrated cell strain and application thereof

Abstract

The invention provides a site-specific integration cell strain screening method and application thereof, and relates to the technical field of biologication.The site-specific integration cell strain screening method comprises the steps that a target gene is integrated into a genetic material of a first cell in a site-specific mode, and a first candidate cell population is formed; the genetic material of the first cell contains and expresses a fluorescence labeling gene, the target gene does not contain the fluorescence labeling gene, and the fluorescence labeling gene can be removed when the target gene is integrated into the genetic material of the first cell at a fixed point; then sorting cells which do not express the fluorescence labeled gene, preparing monoclonal cells and culturing; and sequencing the monoclonal culture cells according to the expression quantity of the target gene coding protein, and selecting the cells of which the expression quantity sequence of the target gene coding protein does not exceed a first threshold value as the screened fixed-point integrated cell strain. The screening method alleviates the problems of complex operation, long screening time and high cost of a site-specific integrated cell strain screening method in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a cell line screening method, and specifically to a site-specific integration cell line screening method and application thereof. Background Art

[0002] Random integration is the most established traditional method for constructing protein expression systems. After entering the cell nucleus, the target gene integrates into the chromosome at random locations. However, cell lines derived from random integration suffer from the problem of poor stability of the resulting clones. Site-directed integration involves precisely inserting a foreign gene into a specific location in the host genome. However, current cell lines derived from site-directed integration still rely on random integration to select single clones. While this method improves stability compared to random integration, the overall process remains cumbersome, time-consuming, and costly.

[0003] Traditional screening of high-expressing monoclonal cell lines is usually performed in the following two ways:

[0004] The first method is to introduce the target gene expression vector into the cells by electroporation, and then divide the cells into a large number of cell pools with the same cell density, that is, to construct minipools. The minipools are then pressure-screened through the resistance to the selection marker on the target gene expression vector, and the minipools with high expression of the target gene are screened for monocloning, and the final high-yield stable monoclonal cell line is found from multiple minipools. This process generally takes about 12 to 14 weeks from electroporation to monoclonal RCB freezing and evaluation. Its advantages are stable monoclonal results and a relatively mature process. Its disadvantages are long screening time, high consumption of consumables and materials, and large manpower and material resources, resulting in high costs.

[0005] The second method is to introduce the target gene expression vector into the cells by electroporation, pressure-screen all the electroporated cells, and then culture the cells after pressure screening until the cell viability recovers to 90%, and clone the cell pool with recovered cell viability. This process from electroporation to monoclonal RCB freezing and evaluation generally takes about 10-12 weeks. Compared with the first process, the advantage is that the process time is shortened and the operation is simpler; the disadvantage is that the throughput used for screening becomes larger, and hundreds of 96-well plates and thousands of monoclonal cells need to be screened, so the overall cost does not decrease; and the different monoclonal cell lines obtained in the end are very different, and a large number of monoclonal cells need to be evaluated to obtain a good and usable monoclonal cell line, which is costly.

[0006] In summary, there is a need for a monoclonal cell line screening method that can save time, is simpler to operate, low-cost, and has more stable results to accelerate the screening of high-expressing monoclonal cell lines obtained through site-directed integration.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for screening site-specific integrated cell lines to alleviate the problems of the prior art of the method for screening site-specific integrated cell lines, such as complex operation, long screening time and high cost.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] In a first aspect, a method for screening a site-specific integrated cell line is provided, the screening method comprising:

[0011] S1. integrating the target gene into the genetic material of the first cell to form a first candidate cell population;

[0012] The genetic material of the first cell contains and expresses a fluorescent marker gene; the target gene does not contain the fluorescent marker gene; the target gene is site-specifically integrated into the genetic material of the first cell and the fluorescent marker gene is removed;

[0013] S2. Cells that do not express the fluorescent marker gene are sorted and prepared into monoclonal cells, and the monoclonal cells are cultured; then, the monoclonal cultured cells are sorted according to the expression level of the target gene-encoded protein, and cells whose expression level of the target gene-encoded protein does not exceed a first threshold are selected as the selected site-specific integrated cell line.

[0014] In an optional embodiment, the target gene is carried by a target gene vector, and the target gene vector does not contain the fluorescent marker gene.

[0015] In an optional embodiment, the target gene and the target gene vector do not express fluorescent molecules; and sorting the cells that do not express the fluorescent marker gene in step S2 is sorting the cells that do not express fluorescence.

[0016] In an optional embodiment, the sorting is performed by flow cytometry, and the flow cytometry sorting conditions include sorting non-fluorescent cells using a single-cell screening mode of a FACS instrument.

[0017] In an optional embodiment, the genetic material of the first cell further contains a screening marker gene, and the screening method further includes subjecting the first candidate cell population to pressure screening after step S1, culturing until the viability of the cells in the first candidate cell population reaches a second threshold, and then performing step S2; the conditions for the pressure screening include inhibiting the growth of cells that do not express the screening marker gene.

[0018] In an optional embodiment, the selection marker gene encodes a protein confers resistance to hygromycin B, zeocin, or puromycin.

[0019] In an optional embodiment, the selection marker gene encodes a protein that is resistant to hygromycin B, and the pressure selection comprises culturing the first candidate cell population under conditions where the concentration of hygromycin B is 250 μg / ml.

[0020] In an optional embodiment, the pressure screening includes screening and culturing the first candidate cell population with 250 μg / ml hygromycin B for 2 days, and after removing the hygromycin B pressure, culturing with normal subculture medium for 3 days.

[0021] In an optional implementation, the second threshold is ≥5%.

[0022] In an optional implementation, the first threshold is ≤36.

[0023] In an optional embodiment, the target gene encodes one or more of an antibody, an enzyme, a hormone, a growth factor and a receptor.

[0024] In an optional embodiment, the fluorescent marker gene includes one or more of a GFP protein gene, an EGFP protein gene, a DsRed protein gene, and an mCherry protein gene. In an optional embodiment, the cell includes a eukaryotic cell.

[0025] In an optional embodiment, the cells include CHO cells, and more preferably include CHO-K1 cells.

[0026] In an optional embodiment, the first cell is a CHO-K1 cell, the integration site of the fluorescent marker gene is within a highly expressed fragment, and the highly expressed fragment comprises the nucleotide sequence shown in SEQ ID No. 1.

[0027] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is the position shown in the annotation information NW_003616785.1:83044 on the CHO-K1 cell.

[0028] In an optional embodiment, the screening method includes using a gene editing system or an integrase system to perform the site-specific integration.

[0029] In an optional embodiment, the enzymes in the integrase system include Cre, Dre, Vika, Bxb1, RDF, FLP, TP901-1、A118、 MR11、TG1、 Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebcuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Hinder, ICleared, Sheen, Mundrea, BxZ2 or or mutants of any of them.

[0030] In an optional embodiment, the integration sites in the integrase system include one or more of a LoxP site, a LoxPL3 site, a LoxP2L site, a LoxFas site, a Lox511 site, a Lox2272 site, a Lox2372 site, a Lox5171 site, a Loxm2 site, a Lox71 site, a Lox66 site, a FRT site, a Bxb1 attP site, and a Bxb1 attB site.

[0031] In an optional embodiment, the first cell is a CHO-K1 cell, the genome of the CHO-K1 cell contains a fluorescent marker protein and a Bxb1 attP site, and the fixed site of the integration site of the fluorescent marker gene is the position shown in the annotation information on the CHO-K1 cell as NW_003616785.1:83044.

[0032] On the second aspect, the application of the site-directed integration cell line screening method of the first aspect in the preparation of proteins or polypeptides is also mentioned.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The site-directed integration cell line screening method provided by the present invention integrates the target gene into the host cell and destroys the original expression of the fluorescent marker in the host cell, and screens cells that do not express the fluorescent marker to obtain monoclones. The time to obtain high-yield monoclones is within 7 weeks, which can be shortened by 3 to 7 weeks compared with the traditional process. The time is short and the cost is low. In addition, the method provided by the present invention is simple to operate and significantly reduces the screening cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 Schematic diagram of the RMCE recombinant plasmid containing target gene 1 constructed in Example 1;

[0037] Figure 2 Schematic diagram of the RMCE recombinant plasmid containing target gene 2 constructed in Example 1;

[0038] Figure 3 Schematic diagram of the Bxb1 recombinase gene plasmid in Example 1;

[0039] Figure 4 The comparison results of target gene expression levels of the screening methods of Example 1 and Comparative Example 1 are shown;

[0040] Figure 5 The results are a comparison of the time taken by the screening methods of Example 1, Comparative Examples 1 and 2. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] definition:

[0043] In this article, "minipool" refers to a cell pool formed by dividing the plasmid-transfected cells into individual containers (in this embodiment, the container is a 96-well plate, but the container is alternative) at a specific density, and then expanding the cells grown in each container (in this embodiment, the container is a 96-well plate, but the container is alternative).

[0044] In this article, "bulk pool" refers to the cell pool formed by pressurizing cells that have been transfected with a plasmid and then waiting for the cell viability to recover.

[0045] "Random integration" herein refers to an integration method in which exogenous genes are randomly inserted into the genome of the host cell.

[0046] In this article, "site-directed integration" refers to the integration method in which a foreign gene is precisely inserted into a specific location in the host genome.

[0047] In this article, "research cell bank" refers to a research cell bank (RCB), which is a population of cells derived from tissues or cells of a single individual, cultured in a specific manner, and characterized as having uniform properties. A cell bank is a collection of containers that store research cells in a specific number of containers under specific conditions. After qualified testing, the stored cells of this cell bank can be used to prepare a master cell bank (MCB), which is then used to prepare working cell banks (WCB).

[0048] In this article, GBB003 cells are CHO-K1 cells carrying attp, attp GA, EGFP and other genes in the middle, and the integration site is NW_003616785.1:83044.

[0049] For definitions of FACS instrument-related parameters and professional terms, please refer to Chinese invention patent application CN201680051655.9, invention title: System and method for adjusting cytometer measurements, application publication number: CN108351287A.

[0050] As used herein, a "target gene vector" refers to a vector that contains and can carry a target gene into a first cell. A vector is a nucleic acid delivery vehicle for inserting a polynucleotide. A vector can be introduced into a host cell through transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell.

[0051] The 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); 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 viruses), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of the present invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRESs), integration sites for enzymes with integration effects, and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0052] In a first aspect, a method for screening a site-directed integration cell line is provided, the screening method comprising steps S1 and S2:

[0053] S1. Integrate the target gene into the genetic material of the first cell to form a first candidate cell population.

[0054] The genetic material of the first cell contains and expresses a fluorescent marker gene; the target gene does not contain the fluorescent marker gene. When the target gene is site-specifically integrated into the genetic material of the first cell, the fluorescent marker gene of the first cell is removed. The genetic material includes endogenous or exogenous genetic material of the first cell. Endogenous genetic material can include the genome of the first cell; exogenous genetic material can include a vector stably expressed in the first cell and integrated with the fluorescent marker gene.

[0055] In an optional embodiment, the target gene encodes one or more of, but is not limited to, antibodies, enzymes, hormones, growth factors, and receptors.

[0056] In an optional embodiment, the protein or polypeptide encoded by the target gene can be a natural protein or polypeptide; or a recombinant protein or polypeptide.

[0057] In an optional embodiment, the fluorescent marker gene includes but is not limited to one or more of GFP protein gene, EGFP protein gene, DsRed protein gene, and mCherry protein gene, preferably EGFP protein gene.

[0058] S2. Cells that do not express the fluorescent marker gene are sorted and prepared into monoclonal cells, and the monoclonal cells are cultured; the monoclonal cultured cells are then sorted according to the expression level of the target gene-encoded protein, and cells whose expression level of the target gene-encoded protein does not exceed a first threshold are selected as the selected fixed-site integrated cell line. The sorting can be performed using any conventional method or device known in the art that can achieve optical analysis and separation of single cells, such as, but not limited to, flow cytometry or a single-cell printer, such as a Namocell, a Cell printer, or a Beacon.

[0059] In an optional embodiment, the target gene is carried by a target gene vector, and the target gene vector contains the target gene; the target gene vector does not contain the fluorescent marker gene, that is, except for the target gene, the rest of the target gene vector does not contain the fluorescent marker gene in the first cell genetic material.

[0060] In an optional embodiment, the target gene or the target gene vector expresses a fluorescent molecule that is different from the genetic material of the first cell. For example, the target gene or the target gene vector expresses mCherry protein, and the first cell expresses EGFP protein, then it expresses red fluorescence. Cells that do not express green fluorescence are positive cells sorted in step S2.

[0061] In an optional embodiment, to further facilitate differentiation, the target gene and the target gene vector do not express a fluorescent molecule. When the target gene is integrated into the genetic material of the first cell and the first cell does not express fluorescence, it indicates that the target gene has been site-specifically integrated into the target location. Therefore, sorting cells that do not express the fluorescent marker gene in step S2 is sorting cells that do not express fluorescence.

[0062] In an optional embodiment, flow cytometry is used for sorting, and the flow cytometry sorting conditions include sorting non-fluorescent cells using a single-cell screening mode of a FACS instrument. Taking the fluorescent marker gene encoding green fluorescent protein as an example, exemplary conditions for sorting cells that do not express fluorescence using flow cytometry include: first, in the single-cell screening mode of FACS, cells in good cell condition are screened out by drawing gates using side scattered light (SSC) and forward scattered light (FSC), and then single cells are screened out by drawing gates using a combination of any two parameters among FSC-A, FSC-H, and FSC-W, or a combination of any two parameters among SSC-A, SSC-H, and SSC-W, and finally, single cells are excited with a 488nm laser to produce green fluorescence, and then the fluorescence intensity in the FITC channel is detected, thereby selecting single non-fluorescent positive cells for sorting.

[0063] In an optional implementation, the first threshold is ≤36, for example, it may be but is not limited to less than or equal to 36, 35, 30, 25, 20, 15 or 10.

[0064] In an optional embodiment, in step S2, cells ranked in the top 36 in terms of expression level of target gene-encoded protein are selected as the site-directed integration cell lines.

[0065] In an optional embodiment, the genetic material of the first cell further contains a screening marker gene, and the screening method further includes subjecting the first candidate cell population to pressure screening after step S1, culturing until the viability of the cells in the first candidate cell population reaches a second threshold, and then performing step S2; the conditions for the pressure screening include inhibiting the growth of cells that do not express the screening marker gene, and the inhibition of cell growth includes but is not limited to death, apoptosis, pyroptosis and / or failure of cells that do not express the screening marker gene to proliferate and produce the next generation of cells.

[0066] In an alternative embodiment, the selection marker gene encodes a protein that confers resistance to hygromycin B, zeocin or puromycin.

[0067] In an optional embodiment, the selection marker gene encodes a protein that is resistant to hygromycin B, and the pressure selection comprises culturing the first candidate cell population under conditions where the concentration of hygromycin B is 250 μg / ml.

[0068] In an optional embodiment, the pressure screening includes screening and culturing the first candidate cell population with 250 μg / ml hygromycin B for 2 days, and after removing the hygromycin B pressure, culturing with normal subculture medium for 3 days.

[0069] In an optional implementation, the second threshold is ≥5%, for example, it may be but is not limited to greater than or equal to 5%, 6%, 7%, 8%, 9% or 10%.

[0070] In an optional embodiment, the first candidate cell population is subjected to pressure screening and cultured until the viability of cells in the first candidate cell population reaches more than 5%.

[0071] In an optional embodiment, the site-specific integration includes using a gene editing system or an integrase system for site-specific integration.

[0072] Exemplary gene editing systems include, but are not limited to, zinc-finger nuclease systems (ZFNs), transcription activator-like effector nuclease systems (TALENs), and clustered regularly interspaced short palindromic repeat sequences (CRISPR).

[0073] The integrase system incorporates the target gene into the genetic material of the first cell by integration, recombination or reverse transcription. Exemplary enzymes in the integrase system that have integration, recombination or reverse transcription functions include but are not limited to Cre, Dre, Vika, Bxb1, RDF, FLP, TP901-1、A118、 MR11、TG1、 Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebcuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Hinder, ICleared, Sheen, Mundrea, BxZ2 or or mutants of any of them.

[0074] Exemplary integration sites in the integrase system include, but are not limited to, one or more of a LoxP site, a LoxPL3 site, a LoxP 2L site, a LoxFas site, a Lox511 site, a Lox2272 site, a Lox2372 site, a Lox5171 site, a Loxm2 site, a Lox71 site, a Lox66 site, a FRT site, a Bxb1 attP site, and a Bxb1 attB site.

[0075] In an optional embodiment, the first cell includes a eukaryotic cell. Exemplary eukaryotic cells include but are not limited to CHO cells, and further preferably include CHO-K1 cells.

[0076] In an optional embodiment, the first cell is a CHO-K1 cell, the integration site of the fluorescent marker gene is within a highly expressed fragment, and the highly expressed fragment comprises the nucleotide sequence shown in SEQ ID No. 1.

[0077] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 11th to 430th base interval of the highly expressed fragment;

[0078] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 21st to 414th base interval of the highly expressed fragment;

[0079] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 38th to 402nd base interval of the highly expressed fragment;

[0080] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 53rd to 389th base interval of the highly expressed fragment;

[0081] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 73rd to 373rd base interval of the highly expressed fragment;

[0082] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 91st to 360th base interval of the highly expressed fragment;

[0083] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 108th to 342nd base interval of the highly expressed fragment;

[0084] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 126th to 326th base interval of the highly expressed fragment;

[0085] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 143rd to 310th base interval of the highly expressed fragment;

[0086] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 160th to 295th base interval of the highly expressed fragment;

[0087] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 178th to 274th base interval of the highly expressed fragment;

[0088] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 194th to 263rd base interval of the highly expressed fragment;

[0089] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 209th to 253rd base interval of the highly expressed fragment;

[0090] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 221st to 242nd base interval of the highly expressed fragment;

[0091] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is any site within the 231st to 240th base interval of the highly expressed fragment;

[0092] In an optional embodiment, the fixed site of the integration site of the fluorescent marker gene is the position shown in the annotation information NW_003616785.1:83044 on the CHO-K1 cell.

[0093] In some specific embodiments, the process of an exemplary site-directed integration cell line screening method is summarized as follows:

[0094] 1. Use electroporation to integrate the target gene into the CHO-K1 specific site NW_003616785.1:83044 with attp and attp GA using Bxb1 recombinase, and replace the green fluorescent marker gene at this site.

[0095] 2. After electroporation, the cells were cultured in subculture medium for 2 days and then subjected to brief pressure screening using 250 μg / mL hygromycin B. The pressure was maintained for 2 days, and then the antibiotic pressure was removed and the cells were cultured in normal subculture medium for 3 days.

[0096] 3. Use a FACS instrument to sort the cells obtained in the previous step. In the single-cell screening mode of FACS, first screen out cells in good cell condition by drawing gates for side scattered light (SSC) and forward scattered light (FSC). The gate range for cells in good cell condition can be obtained by analyzing CHO-K1 cells with a viability greater than 95% under the same voltage conditions. In the SSC-A and FSC-A graphs of CHO-K1 cells with a viability greater than 95%, select the population with a proportion greater than 80% in the image by observing the dot plot. Then, draw different areas for these populations for monoclonal sorting. Compare the survival rate and status of the monoclonal cells after sorting to screen out cells in good cell condition in both SSC and FSC channels. Single cells were then screened by gating on any two combinations of FSC-A, FSC-H, and FSC-W, or any two combinations of SSC-A, SSC-H, and SSC-W. Finally, single cells were excited with a 488nm laser to produce green fluorescence, and the fluorescence intensity in the FITC channel was measured. Single, non-fluorescent positive cells were then sorted into 96-well plates to create monoclonal cells.

[0097] 4. Monoclonal cells are photographed on D0, D1, D2, D3, D7, and D14 (D represents the number of days the monoclonal cells have been cultured) using a VIPS single-cell plating instrument or other monoclonal imaging system. The monoclonal origin is confirmed by observing the continuous growth process of the cells in the photos. After expression screening and expansion, a high-yield monoclonal cell line can be obtained.

[0098] In a second aspect, the use of the site-directed integration cell line screening method described in the first aspect in the preparation of a protein or polypeptide is also provided. The preparation of the protein or polypeptide may optionally include other conventional and optional steps known in the art, including but not limited to one or more steps of expanded culture, enrichment, purification, freezing, and drying, etc., which are not limited by the present invention.

[0099] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0100] The consumables in the following specific embodiments include: BD FACS Flow sheath fluid (BD Biosciences), BD Detergent Solution (BD Biosciences), BD FACS Clean cleaning solution (BD Biosciences), CS&T quality control microspheres (BD Biosciences), Accudrop Beads (BD Biosciences), Neon Resuspension Buffer R (ThermoFisher), a resuspension buffer for cell electroporation; electroporation solution E1 Buffer (ThermoFisher); recovery medium is 80% (v / v) EX-CELL CHO Cloning Medium (Sigma-Aldrich) and 20% (v / v) EX-CELL Advanced CHO Fed-batch Medium (Sigma-Aldrich) supplemented with 1% GlutaMAX (ThermoFisher); expansion medium and subculture medium are both EX-CELL Advanced CHO Fed-batch Medium was supplemented with 1% GlutaMAX (ThermoFisher); pressurized medium was passage medium supplemented with hygromycin at a final concentration of 250 μg / ml; conditioned medium was the supernatant obtained by sterile filtration after inoculating CHO-K1 with passage medium for 1 day; cloning medium was 75% (v / v) EX-CELL CHO Cloning Medium, 20% (v / v) conditioned medium, and 5% (v / v) ClonaCell-CHO ACF Supplement supplemented with 1% GlutaMAX; the basal medium in the fed-batch medium was EX-CELL Advanced CHO Fed-batch Medium supplemented with 1% GlutaMAX (ThermoFisher), and the feed medium was Cell Boost 7a / 7b (HyClone).

[0101] Example 1

[0102] This embodiment mainly includes the following steps:

[0103] (1) Plasmid construction: Sequences containing attB and attB-GA sites and different target protein genes (target gene 1 fragment and target gene 2 fragment) were cloned into different plasmids to construct three recombinant plasmids containing RMCE (recombinase-mediated cassette exchange). The plasmid structures are shown in the schematic diagram. Figure 1 and Figure 2 (Target gene 1 fragment see Figure 1 , target gene 2 fragment see Figure 3 ), the schematic diagram of Bxb1 recombinase gene plasmid is shown in Figure 3 ;

[0104] The recombinant plasmid constructed by target gene 1 has the sequence shown in SEQ ID No. 2, SEQ ID No. 2:

[0105] The recombinant plasmid light chain constructed by target gene 2 has the sequence shown in SEQ ID No. 3, SEQ ID No. 3;

[0106] The heavy chain of the recombinant plasmid constructed by the target gene 2 has the sequence shown in SEQ ID No.4, SEQ ID No.4.

[0107] (2) Resuscitating GBB003 cells. After GBB003 cells have been subcultured for at least two times and the cell doubling time is confirmed to be within 20 ± 2 h, all plasmids constructed in (1) are linearized, and the linear DNA is purified and recovered;

[0108] (3) Take 3×10 6 Transfer the cultured GBB003 cells to a 50 mL centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature, and discard the supernatant.

[0109] (4) Resuspend the cells with 100 μL of R Buffer, a resuspension buffer specifically used for cell electroporation.

[0110] (5) Add 15 μg of the linearized plasmid from step (2) to the resuspended cells from step (4), mix gently, and pipette 50 times to avoid bubbles.

[0111] (6) Turn on the cell electroporator, adjust the parameters, install the motor slot into the electroporator, and add 3 mL of electroporation solution E1Buffer into the slot;

[0112] (7) The cell plasmid suspension prepared in step (5) is sucked into the electroporation gun tip, placed into the electroporation tank, and electroporated;

[0113] (8) Immediately transfer the electroporated cells into a 6-well plate with 2 mL of recovery medium and place in a 37°C, 5% CO2 incubator for overnight culture.

[0114] (9) After 48 hours of electroporation, pressurize the cells with pressurized medium. After 2 days of pressurization, centrifuge at 1000 rpm for 5 minutes at room temperature and discard the supernatant. Switch to passage medium and culture for 3 days until the cell viability recovers to above 5%.

[0115] (10) The cells obtained in the previous step were analyzed using a FACS Melody produced by Becton TM Sorting was performed using the BD Biosciences Melody cell sorter, with the sorting parameters set to Single cell and Flow rate = 1. Non-fluorescent positive cells were sorted into 96-well plates and cultured with cloning medium to complete monoclonal isolation. The specific sorting process is as follows: First, cells in good cell condition are screened by gating on side scattered light (SSC) and forward scattered light (FSC). The gating range for cells in good cell condition can be determined by analyzing CHO-K1 cells with a viability greater than 95% under the same voltage conditions. In the SSC-A and FSC-A graphs of CHO-K1 cells with a viability greater than 95%, the dot plots are used to select populations with a proportion greater than 80% of the image. These populations are then divided into different areas for monoclonal isolation. The survival rate and status of the sorted monoclones are compared to screen out cells in good cell condition based on the performance of the SSC and FSC channels. Single cells were then screened out by drawing gates based on any two combinations of FSC-A, FSC-H, and FSC-W, or any two combinations of SSC-A, SSC-H, and SSC-W. Finally, single cells were excited with a 488nm laser to produce green fluorescence, and the fluorescence intensity in the FITC channel was detected. Thus, single non-fluorescent positive cells were selected and sorted into 96-well plates to make monoclonal cells.

[0116] (11) After 14 days of culture, 40 μL of supernatant was taken from the monoclonal cells that were confirmed to be non-fluorescent under a microscope and the expression level was detected using Elisa or Octet. The top 36 monoclonal cell lines with the highest expression levels were selected and expanded in the expansion medium to form monoclonal cell lines;

[0117] (12) After the monoclonal cell line was expanded to 6 wells, the cells recovered to a cell viability greater than 90% and a viable cell density greater than 1×10 6 cells / mL, the cells were plated at 5×10 5 cells / mL for 6-well batch culture with 2.2 mL of culture medium per well; at the same time, the remaining cells were subcultured in 6-well plates;

[0118] (13) After culturing the cells in the 6-well batch culture for 7 days, the supernatant was collected for expression evaluation, and the top 5 monoclonal cell lines with the highest expression were selected and expanded to shake flasks;

[0119] (14) The monoclonal cell line was cultured in a shake flask stage by fed-batch culture (Advance + 1% Glutamax, i.e., fed-batch culture medium, for the first three days). 3% (v / v) Cell Boost 7a and 0.3% (v / v) Cell Boost 7b (cytiva) were added simultaneously on days 3 and 5. 5% (v / v) Cell Boost 7a and 0.5% (v / v) Cell Boost7b (cytiva) were added simultaneously on days 7, 9, 11, and 13. Sugar was supplemented on days 3, 5, 7, 9, 11, and 13 according to the sugar consumption of the cells and the expression level was evaluated.

[0120] Comparative Example 1 minipool process

[0121] (1) Minipool process The previous process is the same as the process (1) to (8) of this embodiment 1;

[0122] (2) After 48 hours of electroporation, dilute to 3×10 4 cells / mL, evenly distributed into 96-well plates, with 200 μL of culture medium distributed into each 96-well;

[0123] (3) After 14 days of static culture in a 37°C, 5% CO2 incubator, the non-fluorescent cells in the 96-well plate with a large number of non-fluorescent cells were expanded to 24-well plates for culture;

[0124] (4) After the non-fluorescent cell pool is expanded to 6 wells, wait until the cells recover to a cell viability greater than 90% and a viable cell density greater than 1×10 6 cells / mL, the cells were plated at 5×10 5 cells / mL for 6-well batch culture with 2.2 mL of culture medium per well; at the same time, the remaining cells were subcultured in 6-well plates;

[0125] (5) After culturing the cells in the 6-well batch culture for 7 days, the supernatant was collected for expression evaluation, and the top 3 expressing cell pools were selected for expansion and culture to shake flasks;

[0126] (6) When the three cell pools in the shake flask are expanded and the cell viability is restored to greater than 95% and the doubling time is less than 24 hours, the cell pools are cloned using a monoclonal sorting instrument or limiting dilution;

[0127] (7) After 14 days of culture, 40 μL of supernatant from monoclonal cells that have no fluorescence was taken from the plate under a microscope and the expression level was detected using Elisa or Octet. The top 36 monoclonal cell lines with the highest expression were selected and expanded in expansion medium to form monoclonal cell lines;

[0128] (8) After the monoclonal cell line is expanded to 6 wells, wait until the cells recover to a cell viability greater than 90% and a viable cell density greater than 1×10 6 cells / mL, the cells were plated at 5×10 5 cells / mL for 6-well batch culture with 2.2 mL of culture medium per well; at the same time, the remaining cells were subcultured in 6-well plates;

[0129] (9) After culturing the cells in the 6-well batch culture for 7 days, the supernatant was collected for expression evaluation, and the top 5 monoclonal cell lines with the highest expression were selected and expanded to shake flasks;

[0130] (10) The monoclonal cell line was cultured in a shake flask stage by fed-batch culture (Advance + 1% Glutamax, i.e., fed-batch culture medium, for the first three days). 3% (v / v) Cell Boost 7a and 0.3% (v / v) Cell Boost 7b (cytiva) were added simultaneously on days 3 and 5. 5% (v / v) Cell Boost 7a and 0.5% (v / v) Cell Boost7b (cytiva) were added simultaneously on days 7, 9, 11, and 13. Sugar was supplemented on days 3, 5, 7, 9, 11, and 13 according to the sugar consumption of the cells and the expression level was evaluated.

[0131] Comparative Example 2 Bulkpool Process

[0132] (1) Bulkpool process The previous process is the same as the process (1) to (8) of this embodiment 1;

[0133] (2) After 48 hours of electroporation, the cell culture medium was centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended in 2 mL of pressurized medium. The cells were subjected to pressurized screening. After adding antibiotics, the cell growth was checked every 3 to 4 days and the supernatant was centrifuged and discarded. The cells were resuspended in new pressurized medium until the cell viability recovered to 90%;

[0134] (3) The culture is then expanded to a shake flask and subcultured until the cell viability is greater than 95% and the doubling time is less than 24 h. The cell pool is then cloned using a monoclonal sorting instrument or limiting dilution method.

[0135] (4) After 14 days of culture, 40 μL of supernatant was taken from the monoclonal cells that were confirmed to be non-fluorescent under a microscope and the expression level was detected using Elisa or Octet. The top 36 monoclonal cell lines with the highest expression levels were selected and expanded in expansion medium to form monoclonal cell lines;

[0136] (5) After the monoclonal cell line is expanded to 6 wells, wait until the cells recover to a cell viability greater than 90% and a viable cell density greater than 1×10 6cells / mL, the cells were plated at 5×10 5 cells / mL for 6-well batch culture with 2.2 mL of culture medium per well; at the same time, the remaining cells were subcultured in 6-well plates;

[0137] (6) After culturing the cells in the 6-well batch culture for 7 days, the supernatant was collected for expression evaluation, and the top 5 monoclonal cell lines with the highest expression were selected and expanded to shake flasks;

[0138] (7) The monoclonal cell line was cultured in a shake flask stage by fed-batch culture (Advance + 1% Glutamax, i.e., fed-batch culture medium, for the first three days). 3% (v / v) Cell Boost 7a and 0.3% (v / v) Cell Boost 7b (cytiva) were added simultaneously on days 3 and 5. 5% (v / v) Cell Boost 7a and 0.5% (v / v) Cell Boost7b (cytiva) were added simultaneously on days 7, 9, 11, and 13. Sugar was supplemented on days 3, 5, 7, 9, 11, and 13 according to the sugar consumption of the cells and the expression level was evaluated.

[0139] Effect Example 1

[0140] (1) Expression level evaluation: The expression levels of the monoclonal clones of the process of Example 1, the monoclonal clones (random minipool) of the minipool process of Comparative Example 1, and the bulkpool process of Comparative Example 2 were compared. Figure 4 , Figure 4 The results show that the expression level of target gene 1 in Example 1 can reach 1248 mg / L, and the expression level of target gene 2 can reach 5504 mg / L; the expression level of target gene 1 in minipool (Comparative Example 1) can reach 1226 mg / L, and the expression level of target gene 2 can reach 5416 mg / L; the expression level of target gene 1 in bulkpool (Comparative Example 2) can reach 755 mg / L, and the expression level of target gene 2 can reach 3122 mg / L. It can be seen that the expression levels of target genes 1 and 2 in the cell lines screened by the process of Example 1 are higher than those in the cell lines screened by the processes of Comparative Examples 1 and 2, especially significantly higher than those screened by bulkpool.

[0141] (2) Time evaluation: The comparison results of the time used in the process of Example 1, the time used in the minipool process, and the time used in the bulkpool process are shown in Figure 5 . Figure 5The results show that the process of Example 1 only takes 36 days; the minipool (Comparative Example 1) process takes 78 days to screen cell lines with site-directed integration of target gene 1, and 82 days to screen cell lines with site-directed integration of target gene 2; the bulkpool (Comparative Example 2) process takes 63 days to screen cell lines with site-directed integration of target gene 1, and 66 days to screen cell lines with site-directed integration of target gene 2. It can be seen that the time required for the process of Example 1 is significantly less than that required for the processes of Comparative Examples 1 and 2, saving about half the screening time.

[0142] (3) Cost Assessment: The costs of the Example 1 process were compared with those of the minipool process and the bulkpool process. The statistical categories included experimental reagents and consumables, depreciation of experimental equipment (calculated at 10% annual depreciation based on the equipment's useful life), experimental personnel costs, and experimental time costs. The results showed that the cost of the Example 1 method was approximately 60% of the cost of the minipool process (Comparative Example 1) and approximately 69% of the cost of the bulkpool process (Comparative Example 2).

[0143] In summary, the method provided in Example 1 significantly improves the expression of the target gene compared to the bulkpool method (Comparative Example 2), while also saving a lot of time and cost. Compared to the minipool method (Comparative Example 1), the method provided in Example 1 can achieve the gene expression level of the minipool method, but the required time and cost are significantly reduced.

[0144] The preparation of the stable high-fluorescence cells GBB003 used in the above examples and comparative examples mainly includes the following steps:

[0145] (1) The green fluorescent protein gene (EGFP) was used as a marker gene for screening, and the attP sequence was used as a homology arm to construct a recombinant plasmid containing RMCE. After linearizing the constructed plasmid, the linear DNA was purified and recovered. CHO K1 cells were taken, centrifuged, and the supernatant was discarded. The cells were resuspended in 100 μL of R Buffer, a resuspension buffer specifically for cell electroporation, and electroporated three times.

[0146] (2) Pressurization: the pressurization reagent is G418, the pressurization concentration is 800 μg / ml, and the cells are divided into minipools (i.e., 96-well plates) for culture.

[0147] (3) Check the plate after ten days. When a large number of fluorescent cell clusters are observed, they can be enriched. The one-well-to-one-well principle should be followed during enrichment.

[0148] (4) Observe the growth of enriched cells at any time and expand them when the coverage rate reaches more than 50%.

[0149] (5) After expansion to a shake flask, the cells were passaged three times to form a stable cell pool. The formation of monoclonal cells and screening of stable fluorescence were achieved through artificial intelligence. The specific steps are shown in ah.

[0150] a) The cells in the stable cell pool are diluted to a certain concentration and then inoculated into a culture dish containing a semi-solid culture medium. During the inoculation process, the cells are ensured to be approximately evenly distributed in all positions of the culture dish. The cells are allowed to stand for about half an hour to allow the cells to settle to the bottom of the culture dish.

[0151] b) The culture dish is transferred to the electron microscope stage, and the electron microscope performs high-throughput scanning on the cells in the culture dish;

[0152] c) The electron microscope scanned image is uploaded to the server for artificial intelligence image analysis. The analysis process includes monoclonal cell line detection, protein expression level prediction of monoclonal cell lines, protein expression level ranking, and coding and localization of screened high-protein expressing cell lines; the protein expression level prediction of the monoclonal cell line can be based on fluorescence or not. The following step c is the protein expression level prediction not based on fluorescence.

[0153] Step c is based on the detection of monoclonal cell line targets using image processing technology. In this embodiment, the target detection algorithm of YOLOv8 is adopted, and the actual detection effect reaches mAP 94.2%. The target detection model for monoclonal cell lines in step c is consistent with the commonly used deep learning target detection model, so it is not described in detail. Specifically, the cell image needs to be annotated first. In order to improve the prediction accuracy of the algorithm, the bounding boxes of all monoclonal cell lines and adhesion cell lines will be marked and used as the real target bounding box (ground truth) for the loss calculation of the model output. After algorithm training, the model learns the ability to extract the border information of monoclonal cell lines. In the actual application scenario step c, the trained model can predict the border of the monoclonal cell line in the image.

[0154] Step c predicts the fluorescent protein expression level based on monoclonal cell images. In this example, the SqueezeNet deep learning network and the MSE loss function are used. Since the predicted protein expression levels in this project are ranked, the ranking result is the ultimate goal of the algorithm, so the NDCG evaluation standard of the ranking algorithm is used here. The specific calculation formula is as follows:

[0155]

[0156]

[0157] Where IDCG = best-ranked DCG. Specifically in this example, cell imaging predicted expression levels, sorted by predicted expression levels, and compared with the actual fluorescence value sorting results, NDCG = 0.89. The expression level prediction model in this example is described in detail in CN112037862B (application number CN202311058132.5, invention title "A Cell Transfer Method and Cell Transfer System").

[0158] d) The coding and location information of the screened protein high-expressing cell lines are returned to the robot control software;

[0159] The cell codes and positions returned in step d, in this embodiment, return the top 100 cells in terms of predicted expression levels, with the codes ranging from 1 to 100. The position information includes the coordinates in the plane coordinate system relative to the center of the microscope (the depth of the culture medium is not considered for the time being, because all the cells to be selected are deposited to the bottom of the culture dish. In addition, during the photography process, cells that have not settled to the bottom of the culture dish will be out of focus, resulting in blurred cells, and cells with blurred images will also be excluded).

[0160] e) The robot control software automatically operates (or manually assists) the robotic arm to aspirate the screened monoclonal cell lines and transfer them to the designated well plate. This process is repeated until all high-expressing cell lines are transferred. For the suction and transfer process, refer to CN113821287B (application number: CN202111040555.5, invention name “Robot-based cell manipulation task processing method, device, equipment and medium”), CN113403431B (application number: CN202110735660.4, invention name “Robot-based cell liquid collection control method, device, equipment and storage medium”), CN113771030A (application number: CN202111040562.5, invention name “Cell manipulation robot control method, device, equipment and storage medium”), CN113733087B (application number: CN202111039293.0, invention name “Cell manipulation robot control information configuration method, device, equipment and medium”).

[0161] f) After the cell clones in the well plate are cultured to a certain number, they are transferred to a larger well plate for culture, and finally expanded to shake flask culture, and the cell expression level is detected to determine whether the cell line selected by artificial intelligence is a high-yield cell line; step f) in this embodiment is to transfer to a 96-well plate for amplification culture, and then transfer to shake flask culture.

[0162] g) The selected high-yield cell lines are subcultured. Before each generation of cell lines is subcultured, a portion of the sample is diluted and placed in a culture dish, and images are taken using an electron microscope. Images of the cell lines are taken continuously for several generations. Currently, the expression of the selected cells can be predicted by the model learned from the cell morphology using photography, and the expression of the selected cells can also be predicted by fluorescent labeling.

[0163] Step g) Photographing Monoclonal Cells In this example, a Thermo Fisher Scientific M7000 electron microscope was used to photograph the cells.

[0164] h) The collected images of several generations of cell lines are input into an artificial intelligence algorithm to predict the cell line's transgenerational stability based on fluorescence intensity and / or protein expression, and cell lines with high protein expression characteristics that can be stably transgenerated are selected as the final candidate cell lines; the prediction of the cell line's transgenerational stability can be based on fluorescence or on cell morphology rather than fluorescence. The following h) step is the prediction of protein expression based on cell morphology rather than fluorescence.

[0165] Step h) predicting the cell line stability, including but not limited to traditional image recognition algorithms using histograms to extract image features and make predictions, using deep learning neural networks to automatically extract image features and make predictions, and other image-based technical predictions. In this embodiment, a deep learning-based method is used to automatically extract image features of stable cell lines and unstable cell lines and predict their stability, with an accuracy rate of 84% for predictions of different cell lines. See CN114417582A (application number: CN202210010493.1, invention name "Cell line stability prediction method, device, computer equipment and storage medium")

[0166] Wherein, step d) and step e) can be completed manually or automatically by a robot arm. In this embodiment, they are completed automatically by a robot arm.

[0167] (6) CHO-K1 cells were used as control and the seeding density was 5×10 5 cells / ml, inoculate 30 ml of the system, count the cells on the day of inoculation, culture day 3, culture day 5, culture day 7, culture day 9, culture day 11, culture day 13, and culture day 14, and screen out monoclones with a growth rate lower than that of CHO-K1.

[0168] (7) Recover the remaining monoclonal clones and perform stable subculture after recovery. Subculture should be performed every 3 or 4 days. The cell density of the 4-day subculture is 3×10 5 cells / ml, and the cell density after 3 days was 5×10 5cells / ml, and the fluorescence of the cell line was regularly monitored during subculture. The stability of the high-fluorescence cell line was studied for approximately 90 days using the subculture medium. GBB003 cells, which exhibited minimal fluorescence fluctuations and stable cell line growth, were confirmed to be stable high-fluorescence cells. NGS sequencing of the integration site of this cell line revealed the highly expressed fragment sequence located at the integration site as SEQ ID No. 1. Specifically, the annotation information for the integration site on the CHO gene is: NW_003616785.1:83044.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening site-directed integration cell lines, characterized in that: include: S1. integrating the target gene into the genetic material of the first cell to form a first candidate cell population; The genetic material of the first cell contains and expresses a fluorescent marker gene; the target gene does not contain the fluorescent marker gene; The target gene is site-specifically integrated into the genetic material of the first cell and the fluorescent marker gene is removed; S2. Cells that do not express the fluorescent marker gene are sorted and prepared into monoclonal cells, and the monoclonal cells are cultured; then, the monoclonal cultured cells are sorted according to the expression level of the target gene-encoded protein, and cells whose expression level of the target gene-encoded protein does not exceed a first threshold are selected as the selected site-specific integrated cell line.

2. The method for screening site-specific integrated cell lines according to claim 1, wherein: The target gene is carried by a target gene carrier, and the target gene carrier does not contain the fluorescent marker gene; Optionally, the target gene and the target gene vector do not express fluorescent molecules; and sorting the cells that do not express the fluorescent marker gene in step S2 is sorting cells that do not express fluorescence; Optionally, the sorting is performed by flow cytometry, and the flow cytometry sorting conditions include sorting non-fluorescent cells using a single cell screening mode of a FACS instrument; Optionally, the fluorescent marker gene encodes green fluorescent protein, and the flow cytometry sorting conditions include the single-cell screening mode of FACS, in which cells in good cell state are first screened out by drawing gates for side scattered light SSC and forward scattered light FSC, and then single cells are screened out by drawing gates for a combination of any two parameters among FSC-A, FSC-H, and FSC-W, or a combination of any two parameters among SSC-A, SSC-H, and SSC-W, and finally, a 488nm laser is used to excite green fluorescence in the single cell and then the fluorescence intensity in the FITC channel is detected, thereby selecting a single non-fluorescent positive cell for sorting.

3. The method for screening site-specific integrated cell lines according to claim 1, wherein: The genetic material of the first cell also contains a screening marker gene. The screening method further includes subjecting the first candidate cell population to pressure screening after step S1, culturing until the viability of the cells in the first candidate cell population reaches a second threshold, and then performing step S2; the conditions for the pressure screening include inhibiting the growth of cells that do not express the screening marker gene.

4. The method for screening site-specific integrated cell lines according to claim 3, wherein: The selection marker gene encodes a protein resistant to hygromycin B, bleomycin or puromycin; Optionally, the selection marker gene encodes a protein resistant to hygromycin B, and the pressure screening comprises culturing the first candidate cell population at a concentration of 250 μg / ml hygromycin B; Optionally, the pressure screening comprises culturing the first candidate cell population with 250 μg / ml hygromycin B for 2 days, and after removing the hygromycin B pressure, culturing with a normal subculture medium for 3 days; Optionally, the second threshold is ≥5%.

5. The method for screening site-specific integration cell lines according to claim 1, wherein: The first threshold is ≤36.

6. The method for screening site-specific integrated cell lines according to claim 1, wherein: The target gene encodes one or more of antibodies, enzymes, hormones, growth factors and receptors; Optionally, the fluorescent marker gene includes one or more of a GFP protein gene, an EGFP protein gene, a DsRed protein gene and an mCherry protein gene.

7. The method for screening site-specific integrated cell lines according to claim 1, wherein: The cells include eukaryotic cells; Optionally, the cells include CHO cells, further preferably include CHO-K1 cells; Optionally, the first cell is a CHO-K1 cell, the integration site of the fluorescent marker gene is within a highly expressed fragment, and the highly expressed fragment comprises the nucleotide sequence shown in SEQ ID No. 1; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 11th to 430th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 21st to 414th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 38th to 402nd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 53rd to 389th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 73rd to 373rd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 91st to 360th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 108th to 342nd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 126th to 326th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 143rd to 310th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 160th to 295th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 178th to 274th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 194th to 263rd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 209th to 253rd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 221st to 242nd base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is any site within the 231st to 240th base interval of the highly expressed fragment; Optionally, the fixed site of the integration site of the fluorescent marker gene is the position shown in the annotation information NW_003616785.1:83044 on CHO-K1 cells.

8. The method for screening site-specific integration cell lines according to any one of claims 1 to 7, characterized in that: Including using a gene editing system or an integrase system to perform the site-specific integration; Optionally, the enzymes in the integrase system include Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebcuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Hinder, ICleared, Sheen, Mundrea, BxZ2 or φRV, or a mutant of any one of them; Optionally, the integration sites in the integrase system include one or more of a LoxP site, a LoxPL3 site, a LoxP 2L site, a LoxFas site, a Lox511 site, a Lox2272 site, a Lox2372 site, a Lox5171 site, a Loxm2 site, a Lox71 site, a Lox66 site, a FRT site, a Bxb1 attP site, and a Bxb1 attB site.

9. The method for screening site-directed integration cell lines according to claim 8, characterized in that: The first cell is a CHO-K1 cell, the genome of the CHO-K1 cell contains a fluorescent marker protein and a Bxb1 attP site, and the fixed site of the integration site of the fluorescent marker gene is the position shown in the annotation information NW_003616785.1:83044 on the CHO-K1 cell.

10. Use of the method for screening site-directed integration cell lines according to any one of claims 1 to 9 in preparing proteins or polypeptides.

Images