A method for screening stable high expression sites of CHO cells

Abstract

The application belongs to the technical field of genes, and discloses a screening method for stable high expression sites of CHO cells. The method comprises the following steps: 1) extracting DNA of CHO cells integrated with lentivirus; 2) designing specific primers for the 3' LTR region of the lentivirus vector, and performing linear amplification by using single-end DPO primers; 3) purifying PCR products, and adding a linker to the 3' end of the single-stranded DNA after purification; 4) synthesizing double-stranded DNA, and purifying the double-stranded DNA; 5) taking the purified DNA as a template to perform Index PCR; 6) performing sequencing library purification, and then performing sequencing; and 7) filtering the data after sequencing, and performing bioinformatics analysis to obtain information of stable high expression integration sites. The method can be combined with site-specific integration technology to insert an expression exogenous gene at the stable high expression site found, so that a high expression cell line of an exogenous protein can be quickly and efficiently obtained.

Description

Technical Field

[0001] This invention relates to the field of gene technology, specifically to a method for efficient expression of exogenous proteins based on the NC_048604-1 site in the CHO cell genome. Background Technology

[0002] Chinese hamster ovary (CHO) cells are one of the most widely used mammalian cell lines in the biopharmaceutical field. Currently, the annual sales of therapeutic antibodies and proteins in the biopharmaceutical industry have reached more than $300 billion (http: / / www.lamerie.com), of which nearly 84% are produced by Chinese hamster ovary cells, mainly because the recombinant proteins they express have many advantages, such as the ability to perform post-translational modifications similar to human proteins.

[0003] Traditional industrial production methods for constructing recombinant CHO cell lines for expression primarily rely on random integration. This approach involves lengthy screening cycles, and because the insertion site of the target gene cannot be controlled, the spatial environment of the chromosomal region can affect the expression of the exogenous gene, leading to unstable cell line expression and decreased yield over time, which is detrimental to quality control during production. Furthermore, developing high-yield cell lines using existing integration methods remains challenging for proteins that are difficult to express, such as artificial fusion proteins and non-natural bispecific antibodies. These problems ultimately result in extended R&D cycles and increased costs for companies. In recent years, researchers have been dedicated to finding ideal integration sites with high activity and stability for industrial production.

[0004] Currently, various detection and analysis methods are used to find stable high-expression sites, such as Targeted Locus Amplification (TLA) and lentiviral insertion methods. TLA requires the genome of the high-expression cell line and is technically complex, costly, and time-consuming. Meanwhile, existing lentiviral vector insertion site detection and analysis methods, such as chromosome walking, can only identify and verify single-clonal sites, resulting in low throughput, high effort, and uncertain selection of ideal sites. Thermal asymmetric interleaved PCR (TAIL-PCR) and adapter-ligated PCR combined with first-generation sequencing are complex, have low throughput, and low success rates. Whole-genome resequencing involves randomly fragmenting the lentiviral insertion mutant genomic DNA and ligating sequencing adapters to both ends of the fragmented genomic DNA double strands to construct a second-generation sequencing library, which is then sequenced using a second-generation DNA sequencer. While this method can obtain the whole genome sequence information of mutant cells, the sequencing data of flanking sequences at insertion sites typically account for less than 0.01% of the total sequencing data. Furthermore, the larger the genome, the lower the data utilization rate, significantly increasing sequencing costs and making it unsuitable for routine high-throughput identification. Therefore, a high-throughput method for detecting and analyzing integration sites is needed. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a method for efficiently expressing exogenous proteins based on the NC_048604-1 site within the CHO cell genome. This invention utilizes novel detection and analysis methods to identify numerous highly expressed sites within the CHO genome. In the future, site-specific integration technology can be combined to precisely insert exogenous genes into these sites for protein expression, rapidly and efficiently obtaining CHO cell lines with high exogenous protein expression.

[0006] The technical solution of the present invention is as follows:

[0007] A method for efficient expression of exogenous proteins based on lentivirus integration at the NC_048604-1 site in the CHO cell genome and its application, specifically including the following steps:

[0008] (1) Extraction of lentivirus-integrated CHO cell DNA;

[0009] (2) Specific primers LTRI were designed for the 3'LTR region of the lentiviral vector, and linearization was performed using single-end DPO primers;

[0010] (3) Purify the PCR product using AMPure XP magnetic beads, and add adapters to the 3' end of the single-stranded DNA purified in (2) using the VAHTS® ssDNA Library Prep Kit for Illumina.

[0011] (4) Double-stranded DNA was synthesized using Extension Primer and Extension Enzyme Mix, and the double-stranded DNA was purified using AMPure XP magnetic beads;

[0012] (5) Using the purified DNA from (4) as a template, index PCR was performed using primer LTRII. The sequencing library was purified using AMPureXP magnetic beads and then sequenced.

[0013] (6) Filter the sequencing data and perform bioinformatics analysis to obtain information on stable high-expression integration sites;

[0014] Optionally, it also includes (7) expression verification of the screening sites.

[0015] As a preferred embodiment, the specific primer LTRI in step (2) has the nucleotide sequence shown in SEQ ID NO.1.

[0016] As a preferred embodiment, the primer LTRII in step (5) has the nucleotide sequence shown in SEQ ID NO. 2.

[0017] In a preferred embodiment, the data filtering method in step (6) is as follows:

[0018] A. Use a Python script to filter reads containing a specific exogenous sequence (LTR);

[0019] B. A maximum of 6 base mismatches are allowed;

[0020] C. Output the filtered FastQ file;

[0021] D. Use the cutadapt (version 2.8) tool to perform sequence truncation on the filtered fastq file;

[0022] E. By using the parameter -u 66, 66 bases (including primers and foreign sequences) are removed from the 5' end to obtain a clean sequence containing only the target insert fragment.

[0023] In a preferred embodiment, the bioinformatics analysis in step (8) includes:

[0024] A. The bwa mem (Version: 0.7.17-r1188) algorithm is used to align the filtered data with the reference genome, and the data of successfully aligned genomes are output as alignment result files in SAM format;

[0025] B. Use the awk command to extract the chromosome sequence and the alignment position column from the above sam file. This position is the insertion site.

[0026] C. Count the number of identical chromosomes and identical locations, and annotate the relative gene positions.

[0027] The beneficial effects of this invention are as follows: through the research and exploration of this invention and its verification, it is shown that the high-throughput detection and analysis method of this invention can clearly identify the lentiviral insertion sites in the CHO cell genome and quickly identify the insertion location and basic information of the integration site. Attached Figure Description

[0028] Figure 1 : Flowchart of the DPO-NEST method for detecting lentiviral integration sites in CHO cells;

[0029] Figure 2 Analysis of anti-PD1 antibody protein yield in the supernatant of well plate fermentation culture;

[0030] Figure 3 Stability analysis of anti-PD1 antibody protein expression. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Flowchart of the present invention's method for detecting lentiviral integration sites in CHO cells (DPO-NEST) Figure 1 As shown, the following experiment was conducted.

[0033] Example 1: Construction of a high-expression, stable integration lentiviral library:

[0034] A lentiviral vector carrying a green fluorescent tag (GFP) and a glutamine synthase gene (GS) was constructed, and after packaging, a titer of approximately 1 was obtained. 8 10 Lentiviral viruses;

[0035] CHO-S-KOGS cells were infected with a green fluorescent lentivirus with an MOI value less than 0.3 (number of viruses per cell). 96 hours after infection, cells were sorted by flow cytometry. 10 6Cells containing green fluorescence were selected. Lentivirally infected cells were screened using a glutamine-free culture medium under pressure, with selection occurring whenever the cell count reached 8... 10 6 Cell passages were performed at a concentration of 1 / ml, with the initial concentration controlled at 1. 10 6 / ml, after 60 passages, the dominant cell lines were enriched to obtain a stable cell library with high expression of green fluorescent protein, and the top 1% of cells with fluorescence intensity were enriched by flow cytometry.

[0036] Example 2, Lentiviral Integration Site Analysis:

[0037] Information on stable integration sites of highly expressed exogenous proteins was obtained by combining DPO-NEST PCR with next-generation sequencing technology and bioinformatics techniques.

[0038] First, DNA was extracted using Tiangen's cell genomic extraction kit. A 2 μL sample was taken, and the concentration and A260 / A280 of the extracted CHO cell genomic DNA were measured using an ultra-micro UV spectrophotometer. A DNA concentration of 100 ng / μL and an A260 / A280 ratio within the range of 1.9-2.0 were suitable for subsequent PCR experiments.

[0039] Furthermore, specific primers LTRI (AAGCTTGCCTTGAGTGCTTCAAGHHHHHTGTGCCCGT, where H represents hypoxanthine nucleotide) were designed for the 3' LTR region of the lentiviral vector for linear PCR. For each sample, 600 ng of genomic DNA was aliquoted into three tubes (200 ng per tube) to establish a 50 μL PCR reaction system.

[0040] Table 1 shows the linear PCR reaction system.

[0041] ;

[0042] The following reactions were performed on a PCR instrument (Table 2).

[0043] Table 2

[0044] ;

[0045] The above PCR reaction systems were combined to obtain 150 μL of linear amplification product.

[0046] Furthermore, the linear amplification product obtained in the previous step was purified using AMPure XP magnetic beads:

[0047] Equilibrate the AMPure XP magnetic beads to room temperature 30 min in advance and vortex to mix. Add 180 μL of AMPure XP magnetic beads (1.2 ×) to the 150 μL sample above, and pipette 10 times to mix thoroughly. Incubate at room temperature for 5 min to allow DNA to bind to the magnetic beads. Place on a magnetic rack and incubate for 5 min, discarding the supernatant. Wash twice with 200 μL of 80% ethanol, discarding the supernatant and retaining the magnetic beads. Incubate at room temperature for 5 min until the magnetic beads are dry. Remove the sample from the magnetic rack, add 22 μL of nuclease-free water, and pipette 10 times to mix thoroughly. Incubate at room temperature for 2 min. Place on a magnetic rack and incubate for 5 min until the liquid is clear, then transfer 20 μL of supernatant to a new PCR tube.

[0048] Furthermore, adapters were added to the 3' end of the purified single-stranded DNA product obtained in the above steps using the VAHTS® ssDNA Library Prep Kit for Illumina:

[0049] Preheat the PCR instrument: set the heating element to 105℃ and the reaction temperature to 95℃. Heat the purified single-stranded DNA at 95℃ for 2 min, then immediately place it on ice for 2 min. Thaw the 3' Ligation Buffer, 3' Ligation Enzyme Mix, and 3' Adapter at room temperature, mixing thoroughly by inverting before use.

[0050] Prepare the 3' Adapter Ligation premix as shown in Table 3 in a Nuclease-free PCR tube.

[0051] Table 3

[0052] ;

[0053] Gently mix 20 μL of 3' Adapter Ligation premix with the denatured DNA using a pipette, then centrifuge and collect the reaction solution at the bottom of the tube.

[0054] Place the PCR tubes in the PCR instrument and perform the following reactions (Table 4).

[0055] Table 4

[0056] ;

[0057] Furthermore, using the product from the previous step as a template, double-stranded DNA synthesis was completed using Extension Primer and Extension EnzymeMix:

[0058] Thaw the Extension Primer and Extension Enzyme Mix at room temperature, mix thoroughly by inverting the tube, and set aside. Prepare the mixture shown in Table 5 in a Nuclease-free PCR tube.

[0059] Table 5

[0060] ;

[0061] Gently mix the mixture using a pipette, then briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a PCR instrument and perform the following reactions (Table 6).

[0062] Table 6

[0063] ;

[0064] Furthermore, the product from the previous step was purified using AMPure XP magnetic beads:

[0065] Equilibrate the AMPure XP magnetic beads to room temperature 30 min in advance and vortex to mix. Add 96 μL of AMPure XP magnetic beads (1.2 ×) to the 150 μL sample above, and pipette 10 times to mix thoroughly. Incubate at room temperature for 5 min to allow DNA to bind to the magnetic beads. Place on a magnetic rack and incubate for 5 min, discarding the supernatant. Wash twice with 200 μL of 80% ethanol, discarding the supernatant and retaining the magnetic beads. Incubate at room temperature for 5 min until the magnetic beads are dry. Remove the sample from the magnetic rack, add 22 μL of nuclease-free water, and pipette 10 times to mix thoroughly. Incubate at room temperature for 2 min. Place on a magnetic rack and incubate for 5 min until the liquid is clear, then transfer 20 μL of supernatant to a new PCR tube.

[0066] Furthermore, using the product from the previous step as a template, index PCR was performed using primers LTRII (SEQ ID NO. 2: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTGTGTGACTCTGGTAACTAGAGAT, where NNNNNNNN is the known 8 nucleotides used as the P5 index) and P7 Primer (SEQ ID NO. 3: CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC, where NNNNNNNN is the known 8 nucleotides used as the P7 index).

[0067] The reaction is configured according to the following system (Table 7).

[0068] Table 7

[0069] ;

[0070] The following reactions were performed on a PCR instrument (Table 8).

[0071] Table 8

[0072] ;

[0073] Furthermore, the product from the previous step was purified using AMPure XP magnetic beads:

[0074] Equilibrate the AMPure XP magnetic beads to room temperature 30 min in advance and vortex to mix. Add 180 μL of AMPure XP magnetic beads (1.2 ×) to the 45 μL sample above, and pipette 10 times to mix thoroughly. Incubate at room temperature for 5 min to allow DNA to bind to the magnetic beads. Place on a magnetic rack and incubate for 5 min, discarding the supernatant. Wash twice with 200 μL of 80% ethanol, discarding the supernatant and retaining the magnetic beads. Incubate at room temperature for 5 min until the magnetic beads are dry. Remove the sample from the magnetic rack, add 22 μL of nuclease-free water, and pipette 10 times to mix thoroughly. Incubate at room temperature for 2 min. Place on a magnetic rack and incubate for 5 min until the liquid is clear, then transfer 20 μL of supernatant to a new PCR tube.

[0075] First, sequencing was performed on the Illumina Novaseq X platform using a 2×150 bp sequencing strategy. Then, the sequencing data was filtered using the following methods:

[0076] A. Use a Python script to filter reads containing a specific exogenous sequence (LTR);

[0077] B. A maximum of 6 base mismatches are allowed;

[0078] C. Output the filtered FastQ file;

[0079] D. Use the cutadapt (version 2.8) tool to perform sequence truncation on the filtered fastq file;

[0080] E. By using the parameter -u 66, 66 bases (including primers and foreign sequences) are removed from the 5' end to obtain a clean sequence containing only the target insert fragment.

[0081] Furthermore, the filtered data underwent bioinformatics analysis:

[0082] A. Using the bwa mem (Version: 0.7.17-r1188) algorithm, with the specified complex reference genome (complexgenome.fna) as a template, 4 threads (-t 4) are set up to align the processed sequences to the genome, and the data of successfully aligned genomes are output as alignment result files in SAM format;

[0083] B. Use the awk command to extract the chromosome sequence and the alignment position column from the above sam file. This position is the insertion site.

[0084] C. Count the number of identical chromosomes and identical locations, and annotate the relative gene positions.

[0085] The specific bioinformatics analysis results are shown in Table 9.

[0086] Experimental results showed that after exogenous sequence removal, most samples had a read alignment rate (Mapped) greater than 80% and a duplicate read ratio (Duplicates_reads) greater than 90%, which was relatively high. This was due to the amplification cycle during PCR amplification, and PCR product sequencing generally contains a high proportion of duplicate reads. The chimeric read ratio of each sample ranged from 36% to 68%, and there was no significant linear relationship between the number of detected chimeric sites and the number of chimeric reads, indicating that the number of chimeric sites was unrelated to the chimeric read ratio, and the data volume was saturated. The read alignment rate statistics of different samples are shown in Table 9. Analysis revealed that the three samples (M1, M2, M3) generated a total of 1445 integration sites (excluding redundancies and those with more than 100 reads). The integration sites and basic information of some high-read-count samples in M1 are shown in Table 10.

[0087] Table 9. Statistics on the alignment rate of reads from different samples

[0088] ;

[0089] The data includes: original reads (number of original reads used for analysis); duplicate reads (proportion of duplicate reads found during alignment); aligned reads (alignment rate); and chimeric reads (number of reads aligned to both the genome and LTR sequences). These reads are used to analyze integration sites.

[0090] Experimental results showed that DPO-NEST PCR was performed on three CHO cell samples to capture integration sites. First, a Python script was used to screen reads containing specific exogenous sequences (LTRs), allowing a maximum of 6 base mismatches, and outputting a filtered fastq file. The cutadapt (version 2.8) tool was used to truncate the filtered fastq file, removing 66 bases (including primers and exogenous sequences) from the 5' end using the -u 66 parameter, resulting in a clean sequence containing only the target insertion fragment. The bwa mem (Version: 0.7.17-r1188) algorithm was used, with a specified complex genome (complexgenome.fna) as a template, and 4 threads (-t 4) were set to align the processed sequence to the genome, outputting the successfully aligned genome data as a SAM format alignment result file. The awk command was used to extract chromosome columns and aligned position columns from the above SAM file, which are the insertion sites. Then, the number of identical chromosomes and identical positions was counted, and gene relative position annotation was performed. A total of 1445 integration sites were identified, including some sites such as NC_048604-1, NC_048604.1, NC_048598.1, NC_048599.1, NC_048598.1, NW_023276808.1, NC_048595.1, NC_048599.1, NW_023276806.1, NW_023276806.1, NC_048595.1, NC_048604-1, NC_048595.1, NC_048597.1, NC_048604-1, NC_048604.1, NC_048599.1, and NC_048599.1 (Table 10). This invention can effectively capture stable and efficient integration sites in CHO cells.

[0091] Table 10. Integration sites and basic information of some samples

[0092] ;

[0093] Among them, Reads: the number of reads supported for this breakpoint (reads have been deduplicated).

[0094] Test example:

[0095] Using CRISPR / Cas9-mediated site-specific genome editing technology, the anti-PD1 antibody protein gene was specifically integrated into the target sites NC_048604-1, NC_048604.1, NC_048598.1, NC_048599.1, NC_048598.1, NW_023276808.1, NC_048595.1, NC_048599.1, NW_023276806.1, and NW_0232. The expression levels of exogenous proteins at the following sites were selected using this experimental method: NC_048604-1, NC_048595.1, NC_048604-1, NC_048604-1, NC_048604.1, NC_048599.1, NC_048599.1, and the Fer1L4 gene locus, which is reported in the literature as a stable high-expression source. The yield of the NC_048604-1 site was 1.72 times that of the Fer1L4 gene locus. This indicates that the sites selected in this invention can efficiently express exogenous proteins, such as... Figure 2 As shown.

[0096] Simultaneously, the monoclonal cell line integrated into M57 was used at 6 10 5 Cells were seeded in 24-well deep-well plates and cultured continuously for 20 weeks at 37℃, 120 r / min, and 8% CO2. Cell lysate was collected in centrifuge tubes at weeks 4, 8, 12, 16, and 20, and centrifuged at 3000 r / min for 2 min. The supernatant was used to assess protein expression stability. Results showed that all monoclonal cell lines stably expressed anti-PD1 antibody protein, indicating good site-specific expression stability. Figure 3 As shown.

Claims

1. A method for screening stable high-expression sites in CHO cells, characterized in that, Specifically, the steps include the following: (1) Extraction of lentivirus-integrated CHO cell DNA; (2) Specific primers LTRI were designed for the 3'LTR region of the lentiviral vector, and linear amplification was performed using single-end DPO primers; (3) Use magnetic beads to purify PCR products, and add adapters to the 3' end of the purified single-stranded DNA; (4) Perform double-stranded DNA synthesis and purify the double-stranded DNA using magnetic beads; (5) Using the purified double-stranded DNA from (4) above as a template, index PCR was performed using primer LTRII; the sequencing library was purified using magnetic beads and then sequenced. (6) Filter the sequencing data and perform bioinformatics analysis to obtain information on stable high-expression integration sites.

2. The method for screening CHO cell stably highly expressed sites according to claim 1, characterized in that, The specific primer LTRI used in step (2) has the nucleotide sequence shown in SEQ ID NO.

1.

3. The method for screening stable high-expression sites in CHO cells according to claim 1, characterized in that, The primer LTRII used in step (5) has the nucleotide sequence shown in SEQ ID NO.

2.

4. The method for screening stable high-expression sites in CHO cells according to claim 1, characterized in that, The data filtering methods in step (6) are as follows: A. Use a Python script to filter reads containing specific exogenous sequences; B. A maximum of 6 base mismatches are allowed; C. Output the filtered FastQ file; D. Use the cutadapt tool to perform sequence truncation on the filtered fastq file; E. Remove 60-72 bases containing the primer and foreign sequence from the 5' end using the parameter -u 66 to obtain a clean sequence containing only the target insert fragment.

5. The method for screening stable high-expression sites in CHO cells according to claim 1, characterized in that, The bioinformatics analysis in step (6) includes: A. The BWA MEM algorithm is used to align the filtered data with the reference genome, and the data of successfully aligned genomes are output as alignment result files in SAM format; B. Use the awk command to extract the chromosome sequence and the alignment position column from the above sam file. This position is the insertion site. C. Count the number of identical chromosomes and identical locations, and annotate the relative gene positions.

6. The method for screening stable high-expression sites in CHO cells according to claim 1, characterized in that, The lentivirus-integrated CHO cells in step (1) are created by integrating a lentivirus carrying a green fluorescent gene and a glutamine synthase gene into the CHO cell genome.

7. The method for screening CHO cell stably highly expressed sites according to any one of claims 1 to 6, characterized in that, It also includes (7) verifying the expression of the selected high-expression sites.

8. A method for stably expressing a foreign protein, characterized in that, The method includes obtaining a high expression site by the screening method for stable high expression sites in CHO cells as described in any one of claims 1 to 7, integrating a foreign protein gene into the high expression site using site-directed integration technology, and expressing the foreign protein gene.

9. The method as described in claim 8, characterized in that, Site-specific integration technology is achieved using recombinase-based site-specific integration vectors, gene-editing site-specific insertion vectors, or integration vectors based on homology-directed repair.

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