Construction and application of multifunctional CHO (Chinese hamster ovary) cell platform based on fixed-point integration

Abstract

The invention discloses construction of a multifunctional CHO (Chinese hamster ovary) cell platform, which is characterized in that an expression cassette CDbox capable of being recognized by Cre recombinase and Dre recombinase is inserted into a genome hot spot of a Chinese hamster ovary (CHO) cell in a fixed-point manner by utilizing a CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated protein 9) technology. Any sequence with the same recognition sites at the two ends can be replaced by using a Cre / lox system, so that the purpose of quickly inserting genes at the positions is achieved; by using a Dre / rox system, according to a Dre recombinase site contained in a donor sequence, a gene deletion, inversion or replacement function can be performed. Two specific applications of CDbox are implemented, a CHO antibody stable expression strain is rapidly constructed, and a CHO cell surface antibody display and secretion integrated scheme is implemented. The research of CHO cell site specificity integration in the past always focuses on the single function of target gene insertion, and the invention realizes multiple modification of genomes on the basis of traditional recombinase-mediated cassette exchange, and provides wider applicability for protein production and gene function research of mammalian cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell genetic engineering, and particularly relates to the construction and application of a multifunctional CHO cell platform. Background Art

[0002] CHO cells have always been the preferred cell line for expressing recombinant therapeutic proteins. Among the total number of recombinant therapeutic proteins approved in the global market, approximately 70% are produced in CHO cell lines. In the past two decades, with the optimization and development of culture media, production process control, and cell engineering, the production of mammalian cells has achieved a qualitative leap. For example, through fed-batch culture, the CHO protein production has exceeded 13 g / L. However, compared with Escherichia coli, yeast, or insect cell systems, the bottlenecks of mammalian cell systems are the long culture time, low product yield, and phenotypic heterogeneity of transgenic cell clones. Therefore, to meet the growing demand of the biopharmaceutical market, how to engineer productive cells to achieve higher batch productivity in a shorter time, as well as stable product quality and lower production costs, is a research hotspot in the current biopharmaceutical field.

[0003] The generation of traditional CHO cell lines mainly relies on random integration and large-scale screening of high-yield cell lines. Although the processes and hardware facilities have changed significantly during this period, the basic methods remain relatively unchanged. After the target gene enters the cell nucleus, it randomly integrates into the genome, resulting in most cell clones having unfixed transgenic integration sites and copy numbers. Due to the uncontrollability of the insertion position and the inserted gene copy number of the target gene, the obtained randomly integrated cell lines will have problems such as "position effect", and with the extension of the culture passage, the production stability will decline and the expression level will decrease. Therefore, subclonal populations isolated from single cells are characterized by cumbersome cell line characterization according to productivity and other characteristics (including cell viability, glycosylation, stability, etc.) to screen high-yield cell lines. All the development time usually takes 6 to 12 months, which is time-consuming, laborious, and costly.

[0004] To circumvent the variability of random transgene integration and shorten the overall cell line development time, site-specific recombinase technology has been developed to integrate target genes into hotspots of the cell genome. We refer to the double exchange composed of specific site recombinase target sites as recombinase-mediated cassette exchange (RMCE). In gene exchange by the RMCE method, DNA breakage and religation occur only between the corresponding sites, resulting in a protein-producing cell line stably expressing at a fixed site. Since it is difficult to establish recombinase recognition sites in the cell genome in advance as recombinase targets, attempts to apply RMCE to CHO cells have been hard to achieve. With the development of gene editing technology and the discovery of hotspots, a combined method of nuclease homology-mediated RMCE site-specific integration into genomic hotspots has been developed, which can avoid the drawbacks of random integration and generate productive CHO cell lines more quickly. Previous studies on site-specific integration in CHO cells have focused on the single function of target gene insertion. Based on the traditional recombinase-mediated cassette exchange, the present invention uses two recombinases, Cre and Dre, not only to rapidly construct a CHO cell production strain stably expressing foreign genes, but also to achieve multiple modifications of the foreign-expressed genes, providing broader applicability for protein production and gene function research in CHO cells. Summary of the Invention

[0005] The first object of the present invention is to provide a multifunctional CHO cell platform CDbox.

[0006] The second object of the present invention also proposes the application of the multifunctional CHO cell platform CDbox.

[0007] To achieve the first object of the present invention, a recognition expression cassette CDbox that can be recognized by Cre and Dre recombinases is constructed and integrated into the hotspot of the CHO cell genome using CRISPR / Cas9 gene editing technology.

[0008] Among them, the above-mentioned expression cassette CDbox sequentially includes the following functional elements: a promoter, Cre recombinase recognition site sequence 1, a selection marker gene sequence, Cre recombinase recognition site sequence 2, Dre recombinase recognition site sequence, and a polyA tail sequence. Cre recombinase recognition site sequence 1 and Cre recombinase recognition site sequence 2 are two different site sequences recognized by the Cre recombinase, and the order can be interchanged.

[0009] Among them, the above-mentioned promoter includes CMV, EF1α, CAG, or any other strong promoter applicable to eukaryotic cells.

[0010] Among them, the above Cre recombinase recognition sites include loxP, lox2272, lox5171, or any other site recognized by the Cre recombinase; the above Dre recombinase recognition sites include roxP, rox9, rox12, or any other site recognized by the Dre recombinase.

[0011] Among them, the above selection markers include fluorescent proteins, resistance genes, or fusion proteins of fluorescent proteins and resistance genes.

[0012] Among them, the above hot spots of the CHO genome integrating the CDbox expression cassette include but are not limited to hot spots Hipp11, Fer1L4, Rosa26, Hprt, Ywhae, and C12orf35.

[0013] To achieve the second object of the present invention, two applications were implemented. The first is to rapidly construct a stable CHO antibody-expressing cell line, and the second is a scheme for integrating antibody display and secretion on the surface of CHO cells.

[0014] This invention developed a multifunctional CDbox system operating at the genomic hot spots of CHO cells, which can meet different requirements in CHO cell line research applications and cell line development. The CDbox expression cassette corresponds to two recombinase systems: the Cre / lox system and the Dre / rox system. After the CMV promoter of this expression cassette, recognition site 1 of the Cre enzyme is embedded before the ATG, and recognition site 2 of the Cre recombinase and the site recognized by the Dre recombinase are embedded before the BGH and after the terminator. The Cre recombinase can replace any sequence with the same recognition sites at both ends, thereby achieving the purpose of rapidly inserting genes at this position. The function and role of the site recognized by the Dre recombinase depend on the donor sequence. When the donor sequence contains its sites in the same direction, it will exercise a deletion function. When it contains its sites in the opposite direction, it will exercise an inversion function. When it contains its corresponding mutant sites, it will exercise a replacement function.

[0015] Research on site-specific integration of productive mammalian cells such as CHO has been focused on the single function of target gene insertion. The multifunctional CHO cell platform of the present invention only needs to design a simple donor to rapidly achieve site-directed insertion of foreign genes and reduce the troubles caused by random insertion. In addition, multiple modifications of foreign genes can be carried out on this basis, providing broader applicability for protein production and gene function research of mammalian cells. Brief Description of the Drawings

[0016] Figure 1 It is for the multiple applications of the CDbox expression cassette in the CHO cell genome. The CDbox expression cassette can be integrated and perform multiple functions at one or more sites on the CHO cell genome.

[0017] Figure 2 Schematic diagram of site-specific integration of CRISPR / Cas9-mediated CDbox expression cassette. The CDbox expression cassette consists of a CMV promoter, an mCherry gene, a PuroR fusion protein, and BGH poly(a) to form a complete expression system. This expression system contains a Cre recombinase recognition site loxP, a Cre recombinase recognition site lox2272, and a Dre recombinase recognition site roxP, located before the start codon. Through homologous recombination mediated by CRISPR / Cas9 technology, the homologous arms at the ends of the CDbox are connected to the target site and inserted into the H11 site, and the CHO-CDbox cell line is obtained by screening.

[0018] Figure 3 PCR amplification map of different gene elements of the CDbox donor. Lane 1, T2A-PuroR-lox2272-XhoI fragment; Lane 2, HindIII-loxP-mCherry fragment; Lane 3, HindIII-loxP-mCherry-PuroR-lox2272-XhoI fragment; Lane 4, sgRNA1-5’arm; Lane 5, sgRNA1-3’arm; Lane 6, KpnI-CMV-loxP-mCherry-PuroR-lox2272-roxP-BGH-BamHI fragment; Lane 7, 5’arm-CMV-loxP-mCherry-PuroR-lox2272-roxP-BGH-3’arm fragment.

[0019] Figure 4 Junction PCR identification of CHO-CDbox monoclonal positive cell lines. Figure A shows the primer positions and product fragment sizes of 5’ / 3’ Junction PCR. Figure B shows the agarose gel results of 5’ / 3’ Junction PCR.

[0020] Figure 5 Detection of the CDbox copy number in the genomes of candidate cell lines. The qPCR absolute quantification results show that only the CHO-I3 cell line has a single copy (mean ± S.D, n = 3 independent experiments).

[0021] Figure 6 Morphology of CHO-I3 cells under an inverted fluorescence microscope and fluorescence rate detected by flow cytometry.

[0022] Figure 7 Comparison of the proliferation of CHO-CDbox cell lines and CHO-K1 wild-type strains by RTCA instrument.

[0023] Figure 8PCR amplification diagrams of different gene elements of the pMV-HygR-EGFP donor. Lane 1, pMV-loxP-lox2272 plasmid; Lane 2, pMV-loxP-lox2272 plasmid linearized by EcoRI and XhoI; Lane 3, EcoRI-HygR-roxP fragment; Lane 4, roxP-EGFP-XhoI fragment; Lanes 5 and 6, EcoRI-HygR-roxP-EGFP-XhoI fragment;

[0024] Figure 9 Fluorescence expression rates and mean fluorescence intensities (MFI) of CHO-CDHE cells at different passages. Flow cytometry without antibiotics was used to detect the fluorescence expression rates and mean fluorescence intensities (MFI) of CHO-CDHE cells at passages 5, 25, 50, and 75.

[0025] Figure 10 Morphology of CHO-CDHE cells under an inverted fluorescence microscope (left) and fluorescence rates detected by flow cytometry (right) before and after the action of Dre / rox. After the participation of Dre recombinase, some cells in the CHO-CDbox cell pool lost fluorescence.

[0026] Figure 11 Peak diagrams of the key sequences at the H11 locus in CHO-CDHE cells and CHO-CDH monoclonal cells. In CHO-CDH cells, the Dre recombinase deleted the EGFP gene between the two roxP sites.

[0027] Figure 12 PCR amplification diagrams of different gene elements of the pMV-HC-LC-HygR donor. Lane 1, EcoRI-HC-LC-T2A fragment; Lane 2, T2A-HygR-XhoI fragment; Lane 3, EcoRI-HC-LC-HygR-XhoI fragment.

[0028] Figure 13 Junction PCR identification of CHO-CDbox-PAb monoclonal cells. Figure A shows the primer positions and product fragment sizes of 5’ / 3’ Junction PCR. Figure B shows the agarose gel results of 5’ / 3’ Junction PCR.

[0029] Figure 14 Comparison diagram of the proliferation curves of the CHO-CDbox-PAb cell pool and its 8 monoclonal antibody strains.

[0030] Figure 15Comparison chart of antibody secretion of monoclonal antibody strains of different generations. The antibody secretion of the CHO-CDbox-PAb cell pool and its 8 monoclonal antibodies in the 1st, 25th, and 50th generations was detected by the Elisa method (mean ± standard deviation, n = 3 independent experiments).

[0031] Figure 16 PCR amplification map of different gene elements of the pMV-VNAR-Fc-TM-HygR donor. Lane 1, Fc-roxP fragment; Lane 2, roxP-TM fragment; Lane 3, T2A-HygR-XhoI fragment; Lane 4, EcoRI-SP-VNAR fragment; Lane 5, EcoRI-VNAR-Fc-TM-HygR-XhoI fragment;

[0032] Figure 17 Peak map of CDR3 in VNAR of the CHO-CDbox-VFTH cell pool. The CDR3 peak map presents highly complex overlapping peaks, indicating that the CHO-CDbox-VFTH cell pool is a cell library.

[0033] Figure 18 Western blot analysis shows the VNAR-Fc secretion level in the CHO-CDbox-VFTH cell pool. The addition of Dre enzyme significantly increased the secretion of VNAR-Fc in the extracellular medium. L: Cell lysate; M: Extracellular medium. Specific implementation examples

[0034] The concept and technical effects of the present invention will be described in detail below in combination with specific embodiments to fully understand the purpose and effects of the present invention. It should be known that the following specific implementation examples are only used to help those skilled in the art understand the present invention, and they are only a part of the embodiments of the present invention, rather than all the embodiments of the present invention.

[0035] Example 1 Construction of the pX458-sgRNA expression vector targeting the H11 site of CHO-K1 cells

[0036] 1. Obtaining sgRNA targeting the H11 site of CHO-K1 cells

[0037] Hipp11 (H11) in the CHO-K1 genome is located between the conserved genes Eif4enif1 (GeneID: 100762835) and Drg1 (GeneID: 100764489) on chromosome 1, with a total of 5127 base pairs. Using the online website CRISPOR( http: / / crispor.tefor.net / )Design sgRNA targeting the H11 sequence. In this invention, the pX458 (Addgene #48138) vector was used for CRISPR-related experiments. According to the pX458 plasmid, sticky ends were generated by the restriction endonuclease BbsI, so that the sgRNA could be inserted into the plasmid. Therefore, when designing the sgRNA, it is necessary to add the sticky end CACC at the 5' end of its forward oligonucleotide (if the first nucleotide at the 5' end is not G, then G needs to be added first, and then the sticky end CACC), and add the sticky end AAAC at the 3' end of its reverse oligonucleotide. The synthesized sgRNA sequences are shown in Table 1 and were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0038] Table 1 Oligonucleotides of synthesized sgRNA targeting the H11 locus

[0039]

[0040] 2. Construction of the pX458-sgRNA expression vector targeting the H11 locus of CHO-K1 cells

[0041] (1) Annealing of oligonucleotide strands to form sgRNA double-stranded (gDNA): The synthesized oligonucleotide dry powder was dissolved in ddH 2 O to a concentration of 10 μM, mixed according to Table 2, incubated at 95 °C for 5 min in a PCR instrument, taken out and cooled to room temperature naturally, and stored in a 4 °C refrigerator.

[0042] Table 2 Reaction system for annealing of sgRNA oligonucleotide strands

[0043]

[0044] (2) Linearization of the pX458 plasmid: The pX458 plasmid was digested with the restriction endonuclease BbsI alone. The digestion system was according to Table 3, digested in a 37 °C water bath for 3 h, and the digestion degree was examined by 1% agarose gel, and the completely digested linearized plasmid was recovered using a gel extraction kit.

[0045] Table 3 Digestion system of the pX458 plasmid

[0046]

[0047] (3) Ligation of the digestion product and the sgRNA double-strand: The pX458 linearized plasmid and the sgRNA double-strand fragment were mixed at a ratio of 1:3, and the reaction system was prepared according to Table 4, and placed in a 16 °C water bath for overnight ligation.

[0048] Table 4 Reaction system for ligation by T4 ligase

[0049]

[0050] (4) Transformation of DH5α competent cells: Add 10 μL of the ligation product to 100 μL of DH5α competent cells thawed on ice, mix gently, and incubate on ice for 30 min; heat shock in a 42°C water bath for 90 s, then return to ice bath for 2 min; add 500 μL of fresh LB liquid medium, culture at 37°C with shaking at 200 rpm for 1 h; centrifuge at 4000 rpm for 5 min, discard the supernatant, add 100 μL of fresh LB liquid medium, resuspend the cells, and then spread them on an LB solid culture dish containing ampicillin antibiotic. Incubate in an inverted culture incubator at 37°C for 12 h to obtain monoclonal colonies.

[0051] (5) Sequencing identification and plasmid extraction of positive monoclonal: Randomly pick some monoclonal colonies into 1 mL of LB liquid medium, culture at 37°C with shaking at 200 rpm for 4 h, take out 200 μL of the bacterial liquid and send it to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing primer is the universal primer U6. Inoculate the monoclonal bacterial liquid with correct sequencing results into 10 mL of LB liquid medium and culture overnight. Use a plasmid mini-prep kit to extract the plasmid, and store the extracted pX458-sgRNA plasmid in a -20°C refrigerator.

[0052] Example 2 Design and construction of CDbox expression cassette donor targeting H11 locus

[0053] 1. Design of CDbox expression cassette donor targeting H11 locus

[0054] In this example, the main steps for constructing the CDbox donor vector for site-directed integration at the H11 locus mainly include the construction of the H11 homologous arm plasmid pCDNA3.1(+)-H11-arm, the construction of the CDbox expression cassette plasmid pDNA3.1-CDbox, and the insertion of the CDbox expression cassette between the 5'-arm and 3'-arm.

[0055] In this example, the CDbox expression cassette can efficiently express the screening marker protein mCherry-T2A-PuroR through the CMV promoter. The purpose of expressing the puromycin resistance gene is to quickly obtain a stable expression platform cell line through antibiotic screening. The purpose of expressing mCherry is to produce a red fluorescent reporter gene, which can be quickly screened by fluorescence color changes when the gene is exchanged by Cre recombinase in the future. A loxP site that can be recognized by Cre recombinase is added to the 5' end of the start codon ATG sequence of the screening marker mCherry-T2A-PuroR, and a variant site lox2272 that can be recognized by Cre recombinase and a roxP site that can be recognized by Dre recombinase are added to the 3' end of the stop codon TGA. Under the premise of the presence of Cre enzyme, the screening marker mCherry-T2A-PuroR gene sequence can be replaced with any gene with a recombinase recognition site (loxP-Gene-lox2272), thereby achieving the purpose of quickly inserting genes at this position; the role and function of the roxP site depends on the donor sequence. Under the action of Dre recombinase, when the donor sequence contains a roxP site in the same direction, it will perform a deletion effect, when it contains a roxP site in the opposite direction, it will perform an inversion effect, and when it contains another roxP variant site, such as roxp12, it will perform a replacement function. Using this CHO-CDbox platform, we can use the Cre recombinase alone, or in combination with the Dre recombinase, to quickly obtain the target stable cell line, such as Figure 1 shown.

[0056] The Cas9 protein cuts the genomic site targeted by the sgRNA and generates DNA double-strand breaks (DSBs). The same base sequences as the two ends of the DSBs are added to the 5' and 3' ends of the expression cassette, respectively, becoming homologous sequences of the DSBs site. The homologous sequences can be site-specifically integrated into the DSBs position through homologous recombination (HDR) of the cell.

[0057] 2. Construction of CDbox expression cassette donor targeting H11 locus

[0058] First, based on the genomic information of CHO-K1, primers were designed to amplify the 510-bp homologous arm at the 5' end and the 470-bp homologous arm at the 3' end of the sgRNA target site respectively. They were ligated by overlap extension PCR, with BamHI and KpnI restriction sites added in the middle, NdeI and ApaI restriction sites added at both ends respectively, and then recombined into the pcDNA3.1(+) plasmid to obtain the plasmid pcDNA3.1(+)-H11-arm. Through primer design, the mCherry gene sequence containing the loxP site at the 5' end was PCR amplified from the plasmid pcDNA3.1(+)-mCherry constructed in the laboratory, and the T2A-PuroR gene sequence containing the lox2272 site at the 3' end was amplified from the PX459 vector. The two were combined by overlap extension PCR to obtain the linear sequence HindIII-loxP-mCherry-T2A-PuroR-lox2272-XhoI. The linear plasmid pcDNA3.1(+) containing the roxP site was obtained by PCR amplification, with the restriction enzyme sites HindIII and XhoI at both ends respectively. The two linear sequences were digested with HindIII and XhoI and then ligated with T4 ligase to obtain the plasmid pcDNA3.1(+)-CDbox. By PCR amplification, KpnI and BamHI restriction sites were added at both ends of its expression cassette, and it was recombined into the middle of the two homologous arms of the plasmid pcDNA3.1(+)-H11-arm. Primers were designed for the 5' end and 3' end of the homologous arms to obtain the linearized CDbox donor with site-specific integration at the H11 site. The primer sequences are shown in Table 5, the PCR reaction system is shown in Table 6, and the digestion system, T4 ligation enzyme system and other plasmid recombination steps refer to Example 1.

[0059] Table 5 PCR primer sequences for constructing the donor vector

[0060]

[0061]

[0062] Table 6 PCR reaction system

[0063]

[0064] Example 3 Establishment of CHO-CDbox positive monoclonal cell line and site-specific integration analysis

[0065] 1. Resuscitation, medium change, subculture and cryopreservation of CHO-K1 cells

[0066] Cell resuscitation: Prepare warm water at 37°C and complete medium in advance. Take out the CHO-K1 cells from liquid nitrogen, quickly put them into the warm water and stir constantly to thaw them rapidly. Add 4 mL of medium at 37°C to a T25 culture flask, add the thawed cell suspension, gently shake evenly, and place it in a 5% carbon dioxide incubator at 37°C for about 6 h. After the cells adhere to the wall, replace the fresh complete medium.

[0067] Cell medium change and subculture: Observe under an inverted microscope. If the CHO-K1 cells have not filled the culture flask, change the medium. Aspirate the old medium, wash twice with PBS, add 5 mL of fresh medium, and put it back into the carbon dioxide incubator for continued culture. If the confluence of CHO-K1 in the culture flask is about 90%, subculture. Aspirate the old medium, wash three times with PBS, add 0.5 mL of 0.25% trypsin, put it back into the carbon dioxide incubator at 37°C for digestion for 2 - 3 min. After the cell morphology becomes round, add 1 mL of complete medium to terminate digestion, and gently pipette the cells to make them suspended. Transfer the cell suspension to a 1.5 mL centrifuge tube and centrifuge at 800 rpm for 5 min. Remove the supernatant, add 1 mL of complete medium, disperse the cells, transfer one-fourth of the cell suspension to the culture flask for subculture, and add 5 mL of complete medium, then put it back into the carbon dioxide incubator for continued culture.

[0068] Cell cryopreservation: Follow the same steps as subculture. After obtaining the cells by centrifugation, add 1 mL of cryopreservation solution, gently blow and mix the cells evenly, transfer them to cryotubes, label information such as cell name, preserver, and preservation time, place them at 4°C for 0.5 h, -20°C for 2 h in sequence, then transfer them to an ultra-low temperature refrigerator, and transfer them into liquid nitrogen for cryopreservation the next day.

[0069] 2. Establishment of CHO-CDbox positive monoclonal cell line

[0070] Co-transfect pX458-sgRNA and linearized donor into CHO-K1. Add each component according to the system of a 6-well plate. The total DNA amount is 2 μg, which is composed of the PX458-sgRNA plasmid and the linearized donor at a mass ratio of 1:1, and the transfection reagent is 4 μL.

[0071] Screening of puromycin-resistant cell pools: 48 h after transfection, change the medium to the medium containing 10 μg / mL puromycin, change it every two days, and perform 96-well plate monoclonal cell screening after two weeks of culture.

[0072] 96-well plate monoclonal cell screening: Trypsinize and collect the cells that have been screened with puromycin for 2 weeks; count them using a hemocytometer and dilute to 5 cells / mL, then add 0.1 mL of this cell suspension into each well of a 96-well plate. After culturing for 5 days, observe under an inverted microscope and look for monoclonal cell wells, change the medium of the monoclonal cell wells to the medium containing puromycin, and continue to expand the culture of the monoclonal cells until subculture and cryopreservation.

[0073] 3. Site-specific integration verification of CHO-CDbox positive monoclonal cell lines

[0074] After obtaining the monoclonal positive cells expressing red fluorescent protein and having puromycin resistance, perform Junction PCR analysis on the monoclonal cells. For Junction PCR, two pairs of primers need to be designed first. The upstream and downstream primers correspond to the genomic sequence of the H11 site and the inserted gene sequence respectively, and the homologous arms of the inserted sequence need to be avoided: 5’F out Located upstream of the 5‘ homologous arm of H11, 5’R in Located downstream of the 5’ homologous arm of the insertion, 3’F in Located upstream of the 3’ homologous arm of the insertion, 3’R out Located downstream of the 3’ homologous arm of H11. The primer sequences are shown in Table 7.

[0075] Table 7 Primer sequences for Junction PCR

[0076]

[0077] Use the Animal Tissue PCR Kit from TransGen Biotech Co., Ltd., Beijing to obtain the genome. Digest the monoclonal positive cells, take 1 - 5×10 5 cells, centrifuge at 1000 rpm to remove the medium, wash the cells twice with PBS, remove the remaining PBS, add a mixture of AD1 buffer and AD2 buffer (40 μL + 10 μL), incubate at room temperature for 10 min, incubate at 95℃ for 3 min, add 40 μL of AD3 buffer, mix well and directly use it as a template for PCR. The reaction system of Junction PCR is shown in Table 8, and the reaction program is shown in Table 9.

[0078] Table 8 Reaction system of Junction PCR

[0079]

[0080] Table 9 Reaction program of Junction PCR

[0081]

[0082] The Junction PCR products were verified by 1% agarose gel electrophoresis. According to the electrophoresis results, positive monoclonal clones with correct Junction PCR bands were selected and sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing primers were 3’F in and 5’R in , and the monoclonal cells with correct sequencing were the candidate cell lines of CHO-CDbox.

[0083] Example 4 Further examination of CHO-Cbox cell line

[0084] 1. Single-copy verification experiment of CHO-Cbox cell line

[0085] The gene copy number of the stable transfected cell line was calculated by the qPCR absolute quantification method. The plasmid with known copy number was used as the standard product and diluted into 7 concentrations according to the gene copy number concentration gradient. The calculation formula of the gene copy number is:

[0086]

[0087] where C is the concentration of the plasmid (ng / μL), and Length is the size of the plasmid sequence (bp). According to the formula, the plasmid concentration was gradient diluted to 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10、1 copies / μL, and used as the DNA template of the standard curve for qPCR amplification. After the end, the logarithm value of the copy number of the standard product was used as the abscissa, and the measured Ct value was used as the ordinate to draw the standard curve, and the amplification efficiency was calculated according to the standard curve. The Ct value of the cell genome sample was brought into the calculation to obtain the absolute copy number of the target gene in the sample. The upstream primer of qPCR was 5’-CCCACAACGAGGACTACACC-3’, and the downstream primer was 5’-GGGCTTGTACTCGGGTCATTG-3’. The qPCR reaction system was as shown in Table 10, and the reaction program was as shown in Table 11.

[0088] Table 10 qPCR reaction system

[0089]

[0090] Table 11 qPCR reaction program

[0091]

[0092] 2. Detection of fluorescence rate of CHO-CDbox cells by flow cytometry

[0093] In addition to observing the red fluorescence of cells under a fluorescent inverted microscope, flow cytometer (FCM) was used to detect the mCherry red fluorescence rate of cells. The collected target cells were washed twice with PBS, resuspended in 200uL PBS, and analyzed by flow cytometry. The selected channel was Y610 (mCherry).

[0094] 3. Verification of fluorescence rate and proliferation activity of CHO-Cbox cell lines

[0095] Using CHO-K1 wild-type cell line as control, xCELLigence RTCA Instrument (RTCA live cell real-time monitor, Aglient) was used to monitor cell proliferation. After digestion, the cells were diluted with culture medium to 1×10 4 cells / mL, add 150μL to each E-Plate16 well, 3 parallel wells for each sample, add to the matching E-Plate16, operate according to the RTCA live cell real-time monitor, the RTCA live cell real-time monitor will automatically record the cell growth trend.

[0096] Example 4 Using EGFP protein as reporter protein to test the feasibility of CHO-CDbox cell platform

[0097] 1. Construction of the EGFP reporter protein donor vector “pMV-HygR-EGFP”

[0098] In order to evaluate the feasibility of the CHO-CDbox cell platform, we designed and constructed a donor vector "pMV-HygR-EGFP" with EGFP protein as the EGFP reporter protein. Through PCR amplification and overlap extension, the donor vector was fused to the HygR gene and the EGFP gene through the roxP sequence. The loxP site and lox2272 site sequences are located before the start codon and after the stop codon, respectively. The primers used are shown in Table 12, and the construction method refers to the CDbox expression cassette donor construction method in Example 2.

[0099] Table 12 PCR primer sequences used to construct the vector

[0100]

[0101] 2. Study on the stability of reporter protein in CHO-CDbox-HygR-EGFP cell line

[0102] According to the cell transfection method in Example 3, the constructed pMV-HygR-EGFP was transfected into CHO-CDbox cells. After 6 hours, the medium was replaced with a normal medium containing 5 μM of TAT-Cre protein. After 96 hours of transfection, the medium containing 200 μg / mL of hygromycin was replaced for 10 days of screening culture to obtain the CHO-CDbox-HygR-EGFP (abbreviated as CHO-CDHE) cell pool. It was found by flow cytometry analysis that the green fluorescence rate of the cell pool screened by hygromycin reached 99.5%.

[0103] To test the stability of the CHO-CDHE cell pool, continuous passage culture was carried out. Without adding the screening antibiotic (hygromycin), taking one day as one passage, samples of the cells of the 5th generation, 25th generation, 50th generation, and 75th generation were taken for flow cytometry analysis of their fluorescence intensity. From the flow cytometry results, the fluorescence expression rates of the CHO-CDHE cell pool of the 5th generation and 25th generation were close to 100%. By the 50th generation and 75th generation, the percentages of fluorescence-expressing cells only slightly decreased to 96.4% and 94.2%. At the same time, the mean fluorescence intensity (MFI) counted by flow cytometry showed that the MFI of CHO-CDHE did not decrease from the 5th generation to the 75th generation. After two and a half months of passage, 94.2% of the positive clones in the CHO-CDHE cell pool showed very stable green fluorescence expression ability, confirming that the H11 locus still maintained good transcriptional activity and expression stability after RMCE mediated by Cre enzyme.

[0104] 3. Verification of the deletion effect of Dre recombinase on EGFP reporter protein

[0105] The medium of the CHO-CDHE cell pool was replaced with a normal medium containing 5 μM of TAT-Dre protein, and the TAT-Dre recombinase gradually reduced the green fluorescence rate of the CHO-CDHE cell pool. Although there was interference from undegraded EGFP, after 168 hours, the proportion of green fluorescent cells detected by flow analyzer decreased to nearly 65.34%. We took out the cells treated for 120 hours and observed under a fluorescence microscope. Some cells did not emit green fluorescence. Eight monoclonal cells expressing green fluorescence (CHO-CDHE1-8) and eight monoclonal cells not expressing green fluorescence (CHO-CDH1-8) were isolated from the cell pool after Dre enzyme treatment, and their genomes were extracted. The sequence of CDbox was amplified by PCR for sequencing. The results showed that the sequencing results were all the same. In the monoclonal cells of the CHO-CDH series that did not express green fluorescence, the EGFP gene between the two roxP sites was excised.

[0106] Example 5 Rapid construction of a stable cell line expressing Pembrolizumab antibody

[0107] In this example, the heavy and light chain sequences of the humanized anti-PD-1 drug Pembrolizumab published by Merck Sharp & Dohme were used for the rapid construction of a CHO antibody expression strain. The construction process was consistent with the CHO-Cbox cell platform verification experiment. First, the plasmid "pMV-HC-LC-HygR" was constructed. The antibody HC-LC sequence was derived from the plasmid pHR_Gal4UAS_Pembrolizumab (Addgene Plasmid #85434), and the T2A-HygR sequence was derived from the "pMV-EGFP-HygR" plasmid constructed in the verification experiment. The primers used are shown in Table 13, and the construction method refers to the CDbox expression cassette donor construction method in Example 2.

[0108] Table 13 PCR primer sequences for constructing the donor vector

[0109]

[0110] According to the cell transfection method in Example 3, the constructed pMV-HC-LC-HygR was transfected into CHO-CDbox cells. After 6 h, the medium was replaced with normal medium containing 5 μM of TAT-Cre protein. After 96 h of transfection, the medium containing 200 μg / mL of hygromycin was replaced for 10 days of screening culture to obtain the cell pool CHO-CDbox-PAb that stably produces Pembrolizumab. Eight monoclonal cells (CHO-CDbox-PAb-1 to 8) were randomly selected from the CHO-CDbox-PAb cell pool for culture. The genomes of the cell pool and these eight monoclonal cells were identified by Junction PCR. There were no differences in the Junction PCR bands of the eight monoclonal cells and the cell pool. The Junction PCR bands were recovered and sequenced, and the sequencing results were exactly the same. The primer sequences used are shown in Table 14. The proliferation curves of the CHO-CDbox-PAb cell pool and the eight monoclonal cells were compared using an RTCA live cell real-time monitor. The RTCA proliferation curves showed no differences in the cell proliferation rates and trends.

[0111] Table 14 Primer sequences for Junction PCR

[0112]

[0113] The CHO-CDbox-PAb cell pool and its eight monoclonal cell lines were cultured in medium without hygromycin. 2×10 of the first generation, the 6th, 25th, and 50th generations were collected respectively. 5The cells were diluted with 2 mL of medium and seeded in a 6-well plate. After incubation for 72 h, the culture supernatant was collected and analyzed for antibody secretion by ELISA. Generally, the difference in antibody secretion between the monoclonal cell lines and the cell pool was small. The results showed that in the first generation, the antibody secretion of the CHO-CDbox-PAb cell pool was 11.43 - 12.93 μg / mL, the antibody secretion of CHO-CDbox-PAb-5 was the highest, at 13.91 - 14.86 μg / mL, and the antibody secretion of CHO-CDbox-PAb-7 was the lowest, at 10.85 - 11.28 μg / mL. The difference in antibody production between individual monoclonal cell lines was within 25%, and compared with the entire cell pool, the difference was within 12%. Compared with the antibody secretion in the first generation, the antibody secretion of the 50th generation of each monoclonal cell line decreased slightly, and the secretion decrease of the CHO-CDbox-PAb cell pool was 7.32%. The decrease of CHO-CDbox-PAb-6 was the smallest, at 5.27%. The secretion of CHO-CDbox-PAb-4 decreased the most, with the average secretion decreasing from 13.87 μg / mL in the first generation to 12.07 μg / mL, a decrease of 12.98%. From the results of this experiment, it can be concluded that the antibody stable transfection cell lines CHO-Cbox-PAb cell pool screened using the CHO-Cbox cell platform have small differences between individuals, high homogeneity, and high expression stability.

[0114] Example 6 A scheme for realizing the integration of mammalian cell surface antibody display and secretion using the CHO-CDbox cell platform

[0115] This example illustrates a scheme for the integration of CHO-CDbox for cell display and antibody expression. Since CHO-CDbox integrates CDbox at the H11 locus in a single copy, when applied to mammalian cell surface antibody display, it can ensure that each cell displays only one antibody. After the cell completes the display, the Dre recombinase can be used to convert the antibody-displaying cells into antibody-secreting cells.

[0116] We constructed the antibody library donor vector pMV-VNAR-Fc-TM-HygR by PCR amplification and overlap extension. The VNAR in it is the shark nanobody library sequence constructed by members of our laboratory. Its N-terminus contains a signal peptide, which is fused with the human Fc fragment. Subsequently, there are the roxP site and the transmembrane sequence TM from the platelet-derived growth factor receptor (PDGFR), as well as the HygR resistance gene with T2A at the N-terminus. The primers used are shown in Table 15, and the construction method refers to the CDbox expression cassette donor construction method in Example 2.

[0117] Table 15 PCR primer sequences for constructing the donor vector

[0118]

[0119] According to the cell transfection method in Example 3, the constructed pMV-VNAR-Fc-TM-HygR was transfected into CHO-CDbox cells. After 6 h, the medium was replaced with normal medium containing 5 μM of TAT-Cre protein. After 96 h of transfection, the medium containing 200 μg / mL of hygromycin was replaced for 10 days of screening culture to obtain the CHO-CDbox-VNAR-FC-TM-HygR antibody library cell pool (abbreviated as CHO-CDbox-VFTH). The CHO-CDbox-VFTH cells use TM to fix the antibody on the cell membrane for antibody display. In a formal experiment, screening of the target antibody can be carried out, which is not exemplified in this case. We extracted the genomic DNA of CHO-CDbox-VFTH cells for sequencing and found that there were relatively severe overlapping peaks in the CDR3 region of VNAR, indicating that this cell pool group is a cell library.

[0120] 5 μM of TAT-Dre enzyme was added to the cell pool medium. The Dre enzyme can cut off TM and HygR between roxP at the genomic level. Then, VNAR-Fc has no transmembrane sequence and will become a secreted antibody strain under the action of the signal peptide at the N-terminus of VNAR. We took equal amounts of cell culture supernatants before and after TAT-Dre treatment for Western blot detection and found that very obvious antibody bands appeared in the cell pool culture supernatant after TAT-Dre treatment, while the sample before treatment showed very faint bands. Thus, it can be shown that after TAT-Dre treatment, a part of the cells in the CHO-CDbox-VFTH cell pool secreted VNAR-Fc into the supernatant. There was also a very small amount of VNAR-Fc in the cell pool culture supernatant before TAT-Dre treatment, presumably due to natural cleavage of a small amount of roxP and detachment of the antibody fragment from the transmembrane protein during the display process.

[0121] In a normal experiment, we can directly collect the antibodies in the cell culture supernatant after screening through the Dre recombinase step for further antibody affinity research, or isolate the secreted monoclonal cell lines for the next step of the experiment. In either case, the step of reconstructing the cell expression strain can be omitted, saving time.

[0122] The above has made a detailed description of the embodiments of the present invention in conjunction with the accompanying drawings. It should be noted that the present invention is not limited to the above embodiments. For those skilled in the art, without departing from the purpose of the present invention, several improvements and refinements made should also be regarded as the protection scope of the present invention.

Claims

1. Construction of a CHO cell platform based on site-specific integration, Characterized in that, Construct an expression cassette CDbox that can be recognized by Cre and Dre recombinases, and use the CRISPR / Cas9 gene editing technology to integrate it into the hot spots of the CHO cell genome.

2. The expression cassette CDbox according to claim 1, Characterized in that, The expression cassette CDbox sequentially comprises the following functional elements: a promoter, Cre recombinase recognition site sequence 1, a selection marker gene sequence, Cre recombinase recognition site sequence 2, Dre recombinase recognition site sequence, and a polyA tail sequence. Among them, Cre recombinase recognition site sequence 1 and Cre recombinase recognition site sequence 2 are two different site sequences recognized by Cre recombinase, and the order can be interchanged.

3. The expression cassette CDbox according to claims 1 and 2, Characterized in that, The promoter includes CMV, EF1α, CAG or any other strong promoter applicable to eukaryotic cells.

4. The expression cassette CDbox according to claims 1 and 2, Characterized in that, The Cre recombinase recognition site includes loxP, lox2272, lox5171 or any other site recognized by Cre recombinase; the Dre recombinase recognition site includes roxP, rox9, rox12 or any other site recognized by Dre recombinase.

5. The expression cassette CDbox according to claims 1 and 2, Characterized in that, The selection marker includes a fluorescent protein, a resistance gene or a fusion protein of a fluorescent protein and a resistance gene.

6. The integration of the CDbox according to claim 1 into the hot spots of the CHO cell genome includes but is not limited to hot spots Hipp11, Fer1L4, Rosa26, Hprt, Ywhae and C12orf35.

7. Application of the CDbox multifunctional expression cassette according to any one of claims 1-6 in any one of the following: (1) Applied to construct a stable expression strain of foreign genes, and the foreign genes include antibody genes and functional protein genes; (2) Applied to the display and screening of antibodies and other drugs on the surface of CHO cells.

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