A method for efficient expression of exogenous proteins based on the NC_048601.1 site in the CHO cell genome

Abstract

This invention belongs to the field of gene technology and discloses a method for efficient expression of exogenous proteins based on the NC_048601.1 site within the CHO cell genome. The site within the CHO cell genome used for stable protein expression is located within the range of bases 16027000-16033000 of NC_048601.1 in the CHO cell genome, and its nucleotide sequence is shown in SEQ ID NO: 1. This invention introduces different protein genes into a fixed location within the CHO cell genome and achieves stable expression.

Description

Technical Field

[0001] This invention belongs to the field of gene technology, specifically relating to a method for efficient expression of exogenous proteins based on the NC_048601.1 site in the CHO cell genome. Background Technology

[0002] The Chinese hamster ovary (CHO) cell line is the most commonly used mammalian expression host for the industrial production of recombinant therapeutic proteins, with 84% of biotherapeutic drugs produced from mammalian cells using this cell line. Key advantages of CHO cells as a production cell factory include their ability to grow in suspension culture, enabling bioprocesses to be scaled up to 12,000 liters or more in stirred bioreactors. Furthermore, CHO cells grow rapidly in high-performance, chemically defined media, achieving high cell densities and are easily genetically modified for heterologous protein expression. Simultaneously, CHO cells efficiently express recombinant proteins with human-like post-translational modifications and exhibit resistance to human pathogenic viruses, highlighting their superiority over other mammalian expression hosts.

[0003] Transient gene expression is an effective way to rapidly supply recombinant proteins, but establishing stable monoclonal production engineered cell lines remains central to modern bioprocessing research to ensure reproducibility for large-scale production. For the past 40 years, the development of stable cell lines has primarily relied on the uncontrolled, random integration of target protein genes into the host cell genome. This process is highly dependent on chance, thus requiring complex and laborious clone screening and identification procedures, including assessing the growth, productivity, recombinant protein quantity, phenotypic and genetic stability of each clone cell line to select stable and high-performance production cell lines. However, recent years have seen significant advancements in transgenic integration technologies, including transposase-mediated semi-targeted integration systems and recombinase or CRISPR nuclease-mediated targeted integration systems. These new methods have significantly changed how CHO production cell lines are constructed.

[0004] However, to ensure stable high transgene expression through targeted integration, the key is to identify stable high-expression hotspots in the genome and evaluate and verify their expression and stability, so that they can be applied to industrial production, shorten the cell line development cycle and reduce costs, and promote industry development. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an application of a site for the stable expression of exogenous proteins at NC_048601.1 within the genome of CHO cells. This site has clearly defined and fixed information, allowing for the insertion of different exogenous protein genes and their stable expression. Furthermore, achieving site-specific integration of exogenous protein genes at this site can significantly shorten the screening cycle of engineered cell lines and reduce research and development costs.

[0006] This invention provides an application of a site for stable expression of a target protein in CHO cells, wherein the site for stable expression of the target protein is located on chromosome 8 of CHO cells, NC_048601.1, within the range of 16027000-16033000, and its nucleotide sequence is shown in SEQ ID NO: 1. The application is to insert an exogenous target protein gene within this range.

[0007] Preferably, the range is within the range of bases 16028000-16032000, specifically within the range of bases 16029000-16031000;

[0008] Preferably, the gene encoding the target protein that is stably expressed is located in the region between the 5' and 3' homologous arms on the expression vector, wherein the 5' and 3' homologous arms are the upstream and downstream homologous arms of the target sequence that can be recognized by CRISPR / Cas9 technology within the range of 16027000-16033000 bases at the NC_048601.1 gene locus;

[0009] More preferably, the target sequence is 5'NNNNNNNNNNNNNNNNNNNNNGG3', which can be recognized by CRISPR / Cas9 technology within the range of 16027000-16033000 bases near the site. Preferably, the target sequence is 5'-CTTAGTGTCTACCATCTGAGAGG-3'.

[0010] The target protein is one of a polypeptide, a functional protein, an antibody, or a fusion protein, preferably a protein with a molecular weight of less than 160 kDa.

[0011] The present invention also provides an expression vector for expressing a target protein in CHO cells, which is used to insert the coding gene of the target protein into a stable target protein expression site in CHO cells; for example, a site-specific integration vector based on recombinase, a site-directed insertion vector for gene editing, or an integration vector based on homology-directed repair;

[0012] Preferably, the gene encoding the target protein is located in the region between the 5' and 3' homologous arms on the expression vector, wherein the 5' and 3' homologous arms are 750 bp homologous sequences upstream and downstream of the site for stable expression of the target protein, respectively.

[0013] The site at which the target protein is stably expressed is at base 16030687 of NC_048601.1 on chromosome 8 in CHO cells.

[0014] Specifically, the expression vector is suitable for expression in CHO cells; the upstream and downstream homologous sequences are 600-900 bp in length, for example 700-800 bp, such as 750 bp.

[0015] More specifically, the target sequence is designed within 3000 bp upstream and downstream of the 16030687th base of the CHO cell gene NC_048601.1, specifically within the range of 16027000-16033000 bases near the 16030687th base of NC_048601.1.

[0016] The nucleotide sequence of the target sequence is further shown as any one of SEQ ID No: 2 to 16.

[0017] In a specific embodiment, the target protein is one of a polypeptide, a functional protein, an antibody, or a fusion protein, preferably a protein with a molecular weight of less than 160 kDa.

[0018] Furthermore, the expression vector also includes a promoter sequence located upstream of the coding gene of the target protein, wherein the promoter controls the expression of the protein;

[0019] Furthermore, the promoters include, but are not limited to: CMV (a strong mammalian expression promoter derived from cytomegalovirus), EF-1a (elongation factor 1). The promoters include strong mammalian expression promoters derived from simian vacuolating virus 40, SV40 (strong mammalian expression promoter derived from simian vacuolating virus 40), and the artificially constructed combined promoter CAG (composed of cytomegalovirus CMV, early enhancer element, and chicken beta-actin promoter).

[0020] The present invention also provides a CHO recombinant cell line, which includes inserting an exogenous target protein gene at a site that stably expresses the target protein, wherein the site that stably expresses the target protein is in the range of bases 69195000-69199000 on chromosome 8 of CHO cells NC_048601.1;

[0021] Preferably, the expression vector, the sgRNA plasmid corresponding to the target sequence, and the Cas9 plasmid are transfected into CHO cells; more preferably, the pressure selection tag has been knocked out in the CHO cells, for example, the glutamine synthase gene has been knocked out.

[0022] The present invention further provides a method for constructing a CHO recombinant cell line for stably expressing a target protein, comprising the following steps:

[0023] (1) The plasmid vector was transfected into CHO cells with the glutamine synthase gene knocked out by electrotransfection to obtain a recombinant CHO-S-KOGS cell pool.

[0024] The plasmids are, respectively, the expression vector, the sgRNA plasmid corresponding to the target sequence, and the Cas9 plasmid; preferably, the molar ratio of the three plasmids is 1:1:2; the transfection is performed using an electroporator.

[0025] (2) The recombinant CHO-S-KOGS cell pool was cultured in well plates or shake flasks;

[0026] (3) Screening to obtain CHO recombinant cells that stably express the target protein.

[0027] When the glutamine synthase gene has been knocked out in CHO cells, the culture in step (2) is carried out using a culture medium without glutamine, and the collected cell culture supernatant is used for dot blot hybridization detection for screening.

[0028] The present invention further provides a method for stably expressing a target protein, which includes the step of fermenting and culturing CHO recombinant cells stably expressing the target protein obtained by the construction method to produce the target protein, preferably including the step of isolating the produced target protein;

[0029] Preferably, the fermentation culture is a fed-batch fermentation culture, and the glucose is maintained at 3.5-4.5 g / L (specifically 4.0 g / L). Fermentation is stopped when the cell viability reaches 80%.

[0030] The beneficial technical effects of this invention are as follows:

[0031] This invention employs site-directed integration technology to integrate the target gene into a stable, high-expression region of a foreign protein. This not only overcomes the instability of foreign protein expression caused by the "position effect" in random integration but also avoids the time-consuming and laborious process of repeatedly screening multiple rounds of high-expression foreign protein monoclonal clones. This shortens the cycle of constructing biopharmaceutical high-expression engineered cell lines to 1-2 months, reducing research and development time and costs. This invention inserts a foreign protein gene at a specific site in the CHO cell genome, achieving stable high expression. Attached Figure Description

[0032] Figure 1 Fluorescence intensity analysis of lentivirus integrated libraries by flow cytometry screening.

[0033] Figure 2 Cas9 expression plasmid.

[0034] Figure 3 :sgRNA plasmid.

[0035] Figure 4 Donor plasmid.

[0036] Figure 5 :mcherry-ccdb plasmid.

[0037] Figure 6 :ccdb-antiPD1-GS-GFP plasmid.

[0038] Figure 7 Screening and identification of monoclonal cell lines expressing anti-PD1 antibody protein.

[0039] Figure 8 Results of 5' and 3' junction PCR amplification of site-specific integration of the anti-PD1 antibody gene and Sanger sequencing analysis.

[0040] Figure 9 Copy number analysis of anti-PD1 antibody genes integrated at specific sites.

[0041] Figure 10 Analysis of anti-PD1 antibody protein yield in the supernatant of well plate fermentation culture.

[0042] Figure 11 Stability analysis of anti-PD1 antibody protein expression.

[0043] Figure 12 Analysis of anti-PD1 antibody protein yield in the supernatant of shake-flask fed-batch fermentation culture. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0045] Example 1: Construction of a high-expression, stable integration lentiviral library:

[0046] 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;

[0047] 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 6 Cells 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 6Cell 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 bank that highly expresses green fluorescent protein. The top 1% of cells by fluorescence intensity were enriched by flow cytometry to obtain a dominant integrated cell pool that stably and highly expresses exogenous protein. After cell expansion and flow cytometry analysis, the fluorescence intensity was significantly increased. Figure 1 ).

[0048] Example 2, Lentiviral Integration Site Analysis:

[0049] Information on stable integration sites of highly expressed exogenous proteins was obtained by combining nested PCR with next-generation sequencing and bioinformatics techniques.

[0050] First, the DNA of the top 1% of cells enriched above was extracted, and specific primers LTRI were designed for the 3'LTR region of the lentiviral vector. Linear PCR was then performed using single-end DPO primers.

[0051] The PCR products were purified using AMPure XP magnetic beads; adapters were added to the 3' end of the purified single-stranded DNA using the VAHTS® ssDNA Library Prep Kit for Illumina.

[0052] Double-stranded DNA was synthesized using Extension Primer and Extension Enzyme Mix, and the double-stranded DNA was purified using AMPureXP magnetic beads. The purified DNA was then used as a template for index PCR using primer LTRII.

[0053] Sequencing libraries were purified using AMPure XP magnetic beads and then sequenced.

[0054] Data analysis begins with using a Python script to filter reads containing a specific exogenous sequence (LTR), allowing a maximum of 6 base mismatches, and outputting a filtered FastQ file. The `cutadapt` tool (version 2.8) is then 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 insert fragment. The `bwa mem` algorithm (Version: 0.7.17-r1188) is then used, with a specified complex genome (`complexgenome.fna`) as a template, and four threads (`-t 4`) configured to align the processed sequences to the genome. Successfully aligned genome data is output as a SAM format alignment result file. Finally, the `awk` command is used to extract the chromosome sequence and the aligned position sequence from the SAM file; this position is the insertion site. The number of identical chromosomes and positions is then counted, and relative gene position annotations are performed. A total of 1,003,221 reads were detected in the range of nucleotides 16,027,000-1,603,3000 on chromosome 8 of the CHO genome (reference sequence NC_048601.1, nucleotide sequence shown in SEQ ID NO:1). This range belongs to an intragene location. Based on the specific location, the relevant reference sequence was downloaded, and a related expression vector was designed and constructed to verify the stable high expression site of the exogenous protein.

[0055] Example 3: Target sequence selection:

[0056] Based on the high-expression exogenous protein stable sites obtained from the above analysis, anti-PD1 antibody proteins were selected for site-specific integration using CRISPR / Cas9 technology. The first step was to design the target sequence for the site. Based on the selected site range (SEQ ID NO:1), the DeepHF | SpCas9 & Base Editor Efficiency Prediction online prediction system was used to design the sequence sgRNA and select the target sequences with high efficiency.

[0057] The online prediction system scores the editing efficiency of the identified 5'NNNNNNNNNNNNNNNNNNNNNGG 3' target sequence: LOW efficacy (score < 0.56); MEDIUM efficasy (0.56 <= score <= 0.74); HIGH efficacy (score > 0.74).

[0058] The target sequences were selected as follows:

[0059] Target sequence 5'-CTTAGTGTCTACCATCTGAGAGG-3' (SEQ ID No:2), score = 0.69;

[0060] The target sequence 5'-ATGGTAGACACTAAGGGTGTGGG-3' (SEQ ID No: 3) had a score of 0.668.

[0061] Target sequence 5'-GATGGTAGACACTAAGGGTGTGG-3' (SEQ ID No:4), score=0.644;

[0062] The target sequence 5'-AGAACATGCCTAAAAGATGGTGG-3' (SEQ ID No: 5) had a score of 0.68.

[0063] Target sequence 5'-ACTCCCTTCTACTATCCCCATGG-3' (SEQ ID No: 6), score=0.68;

[0064] The target sequence 5'-AGCTGACCTGCAACATCCTGGGG-3' (SEQ ID No: 7) had a score of 0.687.

[0065] The target sequence 5'-ATGTGCTAGGAACCAAACTCAGG-3' (SEQ ID No: 8) had a score of 0.657.

[0066] The target sequence 5'-CCAGGAGACACAGAAGGTACAGG-3' (SEQ ID No: 9) had a score of 0.64.

[0067] Target sequence 5'-TGAGAAGTATCAATATCTAGTGG-3' (SEQ ID No: 10), score=0.65;

[0068] Target sequence 5'-AGAGGAGAATTCTATAAGCAGGG-3' (SEQ ID No: 11), score=0.664;

[0069] The target sequence 5'-TGTGAAGCTGATCACAGCGTTGG-3' (SEQ ID No: 12) had a score of 0.67.

[0070] The target sequence 5'-TCCCAGCATTTGAAAAACAGAGG-3' (SEQ ID No: 13) had a score of 0.66.

[0071] The target sequence 5'-GCATTACAGTGTTTCAGGCAAGG-3' (SEQ ID No: 14) had a score of 0.594.

[0072] Target sequence 5'-TTGTAAGGATTAACAGTTGGGGG-3' (SEQ ID No:15), score=0.64;

[0073] Target sequence 5'-GAGATTAGAAAGGGAAAGGAGGG-3' (SEQ ID No:16), score=0.68;

[0074] Furthermore, the three sequences with the highest predicted editing efficiency were selected as target sequences. The optimal target sequence was selected based on the cleavage efficiency using an in vitro Cas9 digestion kit, as shown in SEQ ID No:2.

[0075] Example 4: Site-specific integration of an anti-PD-1 protein gene carrying the green fluorescent gene (GFP) and the glutamine synthase gene (GS)

[0076] CRISPR / Cas9-mediated site-specific genome editing and homologous recombination were used to precisely integrate the green fluorescent protein gene, GS gene, and anti-PD1 antibody protein gene into target sites. CRISPR / Cas9-mediated homologous recombination requires the construction of sgRNA plasmids and Donor Plasmids, the construction process of which is as follows:

[0077] 1. Construction of sgRNA plasmid

[0078] 1) Synthesize oligonucleotide chains according to the target sequence selected in Example 3:

[0079] SgRNA-F:5'-CACCGCTTAGTGTCTACCATCTGAG-3'(SEQ ID No:17)

[0080] SgRNA-R: 5'-AAACCTCAGATGGTAGACACTAAGC-3' (SEQ ID No: 18).

[0081] 2) Anneal and link the synthesized fragments.

[0082] Reaction system: 1ul of SgRNA-F (10uM), 1ul of SgRNA-R (10uM), 1ul of 10×T4 ligase buffer, 7ul of ddH2O, total volume 10ul.

[0083] PCR instrument: 95℃ for 5 min, then store at 4℃.

[0084] 3) Ligate the annealed oligonucleotide chain to the sgRNA-ccdB plasmid (the sgRNA backbone plasmid already available in the laboratory is ligated to the annealed oligonucleotide chain via the Golden Gate).

[0085] Ligation reaction system: 10 μL annealed fragment, 1 μL (100 ng) sgRNA-ccdB plasmid, 1 μL Bsal, 1 μL 10×T4 ligase buffer, 6 μL ddH2O, total volume 20 μL.

[0086] The reaction procedure is as follows:

[0087] 37 ℃ for 3 min, then 16 ℃ for 3 min, repeat 25 times, then 50 ℃ for 3 min, 80 ℃ for 3 min, and finally 4 ℃ for 3 min.

[0088] 4) Transformation to DH5α competent state;

[0089] 5) Select positive single clones and sequence them using the universal primer sgRNA-CX-F:5'-GCCTATTTCCCATGATTCCTTC-3' (SEQ ID No:19);

[0090] 6) Expand positive monoclonal strains and extract plasmids.

[0091] 2. Donor plasmid construction

[0092] Specific information about the Donor plasmid is as follows: Figure 4 As shown, this plasmid mainly includes the anti-PD1 antibody protein expression gene, GS selection gene, GFP and mCherry positive and negative selection genes, two homologous recombination arm genes (5'arm and 3'arm), and the N20+PAM gene sequence containing the target gene, among other key genes. The specific construction method is as follows:

[0093] 1) The 5' homologous arm fragment and the 3' homologous arm fragment were derived from the genomic DNA template, and the mCherry fragment was derived from the mCherry-ccdb plasmid constructed in the laboratory in the previous stage. Figure 5 PCR was performed using primer design, and the relevant fragments were obtained by gel extraction using a kit.

[0094] 2) The recovered fragment was combined with the ccdb plasmid vector containing genes such as anti-PD1, GFP, and GS, which had been previously constructed in the laboratory. Figure 6 The ligation procedure is the same as the sgRNA plasmid construction procedure described above, except that the restriction endonuclease is replaced with Esp3I instead of Bsa1.

[0095] 3) Select positive single clones, sequence them, and extract plasmids after amplification.

[0096] 3. Screening and identification of single-copy, site-specific integration of NC_048601.1 monoclonal cell lines at base 16030687.

[0097] The constructed sgRNA plasmid ( Figure 3 ) and Donor plasmid ( Figure 4 ) and Cas9 plasmids containing the Cas9 protein ( Figure 2 Three plasmids were co-transfected into CHO-S-KOGS cells cultured at 37°C and 5% CO2, with a molar ratio of 1:1:2. Transfection was performed using an electroporator, and selection was conducted on glutamine-free medium. The Donor donor plasmid contains the N20+PAM sequence of the target gene outside its 5' and 3' arm homologous recombination arms. Therefore, after co-transfection, the Donor plasmid was simultaneously recognized and cleaved into linear fragments, improving integration efficiency. Through homologous recombination, the target gene was integrated at the target site under the selection pressure of glutamine-free medium. Simultaneously, the mCherry in the Donor plasmid served as a negative selection tag, excluding randomly integrated monoclonal cells during selection, reducing the workload of later targeted integration monoclonal cell line selection. Once cell viability stabilized and the proportion of green fluorescence increased, monoclonal cells were sorted using BD flow cytometry, selecting cells expressing green fluorescence but not red fluorescence, and then seeded into 96-well plates.

[0098] After monoclonal cells reached 14 days of growth, cell culture supernatant expressing green fluorescence but not red fluorescence was collected. 3 μL of this supernatant was spotted onto an NC membrane and labeled. The membrane was then dried at 37°C, blocked with 5% skim milk powder for 30 min, washed three times with TBST, incubated with goat anti-rabbit secondary antibody (1 μg / mL) for 1 h, washed three times with TBST, and then developed using ECL. Cell lines expressing and secreting anti-PD1 antibody protein were detected. Based on the dot blot hybridization results of the monoclonal supernatant, a total of 44 positive monoclonal cells were initially screened. Figure 7 );

[0099] Positive monoclonal cells were expanded and cultured. A portion was taken for genomic analysis, and PCR identification was performed using 5' junction PCR, 3' junction PCR, and Sanger sequencing. Figure 8 Nine monoclonal cell lines with single-copy integration of the anti-PD1 gene were screened out through copy number identification. Figure 9 ).

[0100] Expand the monoclonal cell lines obtained above.

[0101] Test example:

[0102] The expression of anti-PD1 protein in the monoclonal cell lines obtained in Example 4 was detected using the Octet molecular interaction instrument.

[0103] The nine monoclonal cell lines obtained in Example 4 were subjected to plate fermentation at a ratio of 2... 10 5 Cells were initially seeded at an initial concentration of 0.9 μg / ml and fermented for 6 days. The supernatant was collected, and the expression level of anti-PD1 antibody protein in the fermentation broth was detected using an Octet molecular interaction analyzer and a Protein A sensor. The results showed that the expression level of single-copy integrin ranged from 0.9 to 45.6 μg / ml. Figure 10 The average value was 12.4 μg / ml.

[0104] Four monoclonal cell lines (M44-3, M44-6, M44-21, and M44-56) were selected for 20 consecutive weeks of plate fermentation. Protein expression stability was assessed at weeks 4, 8, 12, 16, and 20. The results showed that all monoclonal cell lines stably expressed anti-PD1 antibody protein, indicating good expression stability at this site. Figure 11 As shown.

[0105] Three monoclonal strains were selected for fed-batch shake-flask fermentation culture every other day. The initial cell concentration was 6 on day 0. 10 5 / ml inoculation, fed culture began on the fourth day. EmCDCHO101 was fed culture medium A at 4% volume, and EmCDCHO101 was fed culture medium B at 0.4% volume. Glucose was maintained at 4g / L. When cell viability reached 80%, cell supernatant was collected. The expression level of anti-PD1 antibody protein in the fermentation broth was detected using a Protein A sensor. The results showed that the expression level of the single-copy integrated anti-PD1 gene at this site in fed shake flask culture ranged from 183.66-455.11 μg / ml, with an average of 323.72 μg / ml. Figure 12 As shown.

[0106] In this invention, the CRISPR / Cas9-mediated genome editing technology is mainly used for site-specific integration. Therefore, target sequences are designed mainly for upstream and downstream sequences within 6000 bp. The 15 target sequences screened in Example 3 of this invention cover most of the upstream and downstream sequences within 6000 bp of this invention. The range of bases 16027000-16033000 in the CHO cell gene NC_048601.1 of this invention can successfully construct stable expression cell lines with site-specific integration and can stably express the target protein.

[0107] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. Use of a site in a CHO cell stably expressing a target protein for stably expressing a target protein, characterized in that, The site for stably expressing the target protein is the chromosome 8 in CHO cells, according to the reference sequence NC_048601.1, within the range of 16027000-16033000 bases of the nucleotide sequence, and the application is to insert the exogenous target protein gene within the range.

2. Use according to claim 1, wherein The site for stably expressing the target protein is the chromosome 8 in CHO cells, according to the reference sequence NC_048601.1, at the 16030687 base within the range of 16027000-16033000 bases of the nucleotide sequence.

3. The use according to claim 1, wherein The target sequence within the range of 16027000-16033000 bases of the nucleotide sequence that can be recognized by the CRISPR / Cas9 technology is 5'-CTTAGTGTCTACCATCTGAGAGG-3'.

4. The use according to claim 1, wherein The target protein is a protein with a molecular weight less than 160 kDa.

5. The use according to claim 4, wherein the compound is ###0002### The target protein is a polypeptide.

6. The use according to claim 4, wherein the compound is ###0002### The target protein is a functional protein.

7. The use according to claim 4, wherein the compound is ###0002### The target protein is a fusion protein.

8. An expression vector for expressing a target protein in CHO cells, characterized by, It is an expression vector for the site for stably expressing the target protein into which the coding gene of the target protein is inserted. The site for stably expressing the target protein is the chromosome 8 in CHO cells, according to the reference sequence NC_048601.1, within the range of 16027000-16033000 bases of the nucleotide sequence.

9. The expression vector of claim 8, wherein, It is a recombinase-based site-specific integration vector, a gene editing site-specific insertion vector, or a homology-directed repair-based integration vector.

10. The expression vector of claim 8, wherein, The coding gene of the stably expressed target protein is located in the region between the 5' homologous arm and the 3' homologous arm on the expression vector, and the 5' homologous arm and the 3' homologous arm are respectively the upstream and downstream homologous arms of 5'-CTTAGTGTCTACCATCTGAGAGG-3' that can be recognized by the CRISPR / Cas9 technology.

11. The expression vector of claim 8, wherein The expression vector further comprises a promoter sequence upstream of the coding gene of the target protein, and the promoter controls the expression of the protein; the promoter is selected from CMV, EF-1a, SV40, or a combination promoter CAG.

12. A CHO recombinant cell line, characterized in that, It comprises inserting the exogenous target protein gene at the site for stably expressing the target protein, which is the chromosome 8 in CHO cells, according to the reference sequence NC_048601.1, at the 16030687 base of the nucleotide sequence.

13. The CHO recombinant cell line according to claim 12, wherein, It is obtained by integrating the target protein gene into the chromosome 8 of CHO cells at the 16030687 base of the nucleotide sequence through the expression vector of any one of claims 8 to 11.

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