A method for high-efficiency expression of exogenous proteins based on NC_048599.1 site in CHO cell genome

Abstract

The application belongs to the technical field of genes, and discloses a method for efficiently expressing exogenous proteins based on a NC_048599.1 site in a CHO cell genome. The site for stably expressing proteins in the CHO cell genome is located in the range of 69195000-69199000 of the CHO cell gene NC_048599.1 shown in the sequence of SEQ ID NO:1. In the fixed position of the CHO cell genome, different protein genes are introduced and stably expressed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic technology, and particularly relates to a method for efficiently expressing exogenous proteins based on NC_048599.1 site in CHO cell genome. BACKGROUND

[0002] Biopharmaceuticals, as one of the most leading and strategic industries in the world, have become a major pillar in the biopharmaceutical industry, and have achieved rapid development in recent years. At present, in the industrial production of biopharmaceuticals, the annual sales of therapeutic antibodies and proteins have reached more than 300 billion US dollars (http: / / www.lamerie.com), of which nearly 84% are produced by Chinese hamster ovary (CHO) cells, mainly due to their strong adaptability, good scalability, and similar post-translational modification characteristics to human proteins. Although it has achieved success in industrial applications, it still faces challenges in achieving consistency and high levels of transgenic expression.

[0003] At present, the main method for constructing recombinant CHO cell strains for expression and production in industrial production is through random integration. This method has a long screening cycle, and since the insertion site of the target gene cannot be controlled, the chromosomal region space environment will affect the expression of the exogenous gene, resulting in unstable expression of the obtained cell strain. This makes it necessary to carry out a large amount of cloning and screening during the research and development process, thus leading to a research and development bottleneck. This variability is due to the influence of genomic sites on transgenic behavior, in which local chromatin structure and epigenetic regulation factors will affect the stability and production efficiency of expression.

[0004] To address these challenges, targeted site integration strategies have become a promising solution, but the CHO cell genome is large, and the stable integration site needs to be mined and evaluated. Therefore, it is necessary to find an ideal integration site with high activity and strong stability and evaluate its expression and stability before it can be applied to industrial production. SUMMARY

[0005] To solve the above technical problems, the present application provides a CHO cell genome NC_048599.1 site for stable expression of exogenous proteins, which has clear and fixed site information, can insert different exogenous protein genes, and can perform stable expression. At the same time, the implementation of exogenous protein gene site integration at this site can greatly shorten the screening cycle of engineering cell strains and reduce research and development costs.

[0006] The application provides a site for stably expressing a target protein in CHO cells, which is a nucleotide sequence on chromosome 6 in CHO cells, and the nucleotide sequence is a NC_048599.1 gene site in the range of 69195000-69199000 bases, and the application is inserting an exogenous target protein gene into the site.

[0007] Preferably, the range is in the range of 69196000-69198000, and specifically in the range of 69196500-69197500.

[0008] Preferably, the coding gene of the 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 the target sequence that can be recognized by the CRISPR / Cas9 technology in the range of 69195000-69199000 bases of the NC_048599.1 gene site.

[0009] Further preferably, the 5' NNNNNNNNNNNNNNNNNNNNNNGG 3' in the range of 69195000-69199000 bases near the site can be recognized by the CRISPR / Cas9 technology as a target sequence, and preferably, the target sequence is 5'-ATGTGTGGATAATATAGTAATGG-3'.

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

[0011] The application also provides an expression vector for expressing a target protein in CHO cells, which is an expression vector for inserting the coding gene of the target protein into the site for stably expressing the target protein in CHO cells; for example, a vector for gene editing or a vector for homologous recombination;

[0012] Preferably, the coding gene of the 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 sequences with a length of 750 bp of the site for stably expressing the target protein.

[0013] The site for stably expressing the target protein is NC_048599.1 at the 69197141th base on chromosome 6 in CHO cells.

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

[0015] More specifically, the target sequence is designed as the sequence within 2000 bp upstream and downstream of base 69197141 of CHO cell gene NC_048599.1, specifically within the range of 69195000-69199000 bases near base 69197141 of NC_048599.1.

[0016] Further, the target sequence is 5'-TATCCCAAGTGAGACATGTGTGG-3';

[0017] The target sequence is 5'-ATGTGTGGATAATATAGTAATGG-3';

[0018] The target sequence is 5'-TCGTTTTATGGATAAAGCAATGG-3';

[0019] The target sequence is 5'-TCTATACCAGGTGTTTCAAGTGG-3';

[0020] The target sequence is 5'-AAACCCAGGACCATTGATTGAGG-3';

[0021] The target sequence is 5'-GCTTTCAGTTACAATTGTAGTGG-3';

[0022] The target sequence is 5'-AGCTTCTCTAAAAAAAGGACTGG-3';

[0023] The target sequence is 5'-AGCTACCTTCTGATGACAGAGGG-3';

[0024] The target sequence is 5'-AAATAGCCTATAGGGAGCAGAGG-3';

[0025] The target sequence is 5'-GTGGAATACTACATGCGCTTAGG-3';

[0026] The target sequence is 5'-GGTTGATACAGGGTTTCAGTGGG-3';

[0027] The target sequence is 5'-AGCAATTCTGTCACTAGTGAGGG-3';

[0028] the target sequence is 5'-CTCTGGTCCTTTGCAGATGAAGG-3';

[0029] the target sequence is 5'-ATTTAGGGAGATAAAGGTTGGGG-3';

[0030] the target sequence is 5'-GCTGAAAGGAGTTGAATGAAGGG-3'.

[0031] In the embodiment, the target protein is one of polypeptide, functional protein, antibody or fusion protein, preferably, the molecular weight of the protein is less than 160 kDa.

[0032] Further, 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.

[0033] Further, the promoter includes but is not limited to: further, the promoter includes but is not limited to: CMV (strong mammalian expression promoter derived from cytomegalovirus), EF-1a (strong mammalian expression promoter derived from elongation factor 1 , SV40 (strong mammalian expression promoter derived from simian vacuole virus 40), and the combination of artificial construction CAG (consisting of cytomegalovirus CMV, early enhancer element and chicken beta-actin promoter).

[0034] The application also provides a CHO recombinant cell line, which comprises an exogenous target protein gene inserted at a site stably expressing a target protein, and the site stably expressing a target protein is in the range of 69195000-69199000 bases of NC_048599.1 on chromosome 6 in CHO cells.

[0035] Preferably, the CHO cell is obtained by the expression vector, sgRNA plasmid corresponding to the target sequence and Cas9 plasmid; more preferably, the CHO cell has knocked out a pressure screening tag, for example, the glutamine synthetase gene is knocked out.

[0036] The application further provides a construction method of a CHO recombinant cell line for stably expressing a target protein, which comprises the following steps:

[0037] (1) The plasmid vector is transfected into CHO cells in which the glutamine synthetase gene has been knocked out by using the method of electroporation, to obtain a recombinant CHO-S-KOGS cell pool;

[0038] The plasmids are respectively the expression vector, the sgRNA plasmid corresponding to the target sequence and the Cas9 plasmid; preferably, the particle molar ratio of the three plasmids is 1:1:2; and the transfection is performed by using an electrotransformation instrument.

[0039] (2) culturing the recombinant CHO-S-KOGS cell pool on a plate or in a shake flask;

[0040] (3) screening to obtain the CHO recombinant cell stably expressing the target protein.

[0041] For the CHO cell in which the glutamine synthetase gene has been knocked out, the culture in step (2) is performed by using a culture medium without glutamine, and the collected cell culture supernatant is used for spot hybridization detection for screening.

[0042] The application further provides a method for stably expressing a target protein, which comprises the steps of fermentatively culturing the CHO recombinant cell stably expressing the target protein obtained by the construction method to produce the target protein, and optionally isolating the produced target protein.

[0043] Preferably, the fermentative culture is performed by using fed-batch fermentation culture, and the glucose is maintained at 3.5-4.5 g / L (specifically 4.0 g / L), and the fermentative culture is ended when the cell viability is 80%.

[0044] The application has the beneficial technical effects that:

[0045] The application uses the site-specific integration technology to site-specifically integrate the target gene into a stable high-expression region, thereby not only overcoming the problem of unstable expression of an exogenous protein caused by "position effect" due to random integration, but also avoiding the time-consuming and laborious repeated screening work of repeatedly screening a high-expression exogenous protein monoclonal, so that the period of rationally constructing a biopharmaceutical high-expression engineering cell strain is shortened to 1-2 months, and the research and development time and cost are reduced.

[0046] The application inserts an exogenous protein gene into a site-specific position in a CHO cell genome to realize stable high expression of the exogenous protein. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 : flow cytometry screening of lentivirus integration library fluorescence intensity analysis.

[0048] Figure 2 : Cas9 expression plasmid.

[0049] Figure 3 : sgRNA plasmid.

[0050] Figure 4 : donor plasmid.

[0051] Figure 5 : mcherry-ccdb plasmid.

[0052] Figure 6 : ccdb-antiPD1-GS-GFP plasmid.

[0053] Figure 7 : Screening and identification of monoclonal cell strains expressing anti-PD1 antibody protein.

[0054] Figure 8 : 5' and 3' junction PCR amplification results of site-specific integration of anti-PD1 antibody gene and Sanger sequencing analysis.

[0055] Figure 9 : Copy number analysis of site-specific integration of anti-PD1 antibody gene.

[0056] Figure 10 : Analysis of anti-PD1 protein yield in well plate fermentation supernatant.

[0057] Figure 11 : Analysis of anti-PD1 protein expression stability.

[0058] Figure 12 : Analysis of anti-PD1 protein yield in shake flask fed-batch fermentation supernatant. DETAILED DESCRIPTION

[0059] The present application will be described in detail below with reference to the accompanying drawings and examples.

[0060] Example 1, construction of high expression and stable integration lentivirus library:

[0061] A lentivirus vector with a green fluorescent tag (GFP) and glutamine synthetase gene (GS) was constructed, and by packaging, a lentivirus with a titer of about 1 8 10 was obtained;

[0062] CHO-S-KOGS cells were infected with green fluorescent lentivirus with an MOI value of less than 0.3 (the number of viruses corresponding to each cell). After 96 hours of infection, 1 10 6 green fluorescent cells were sorted by flow cytometry. The lentivirus infected cells were screened, and a culture medium without glutamine was used for pressure screening. Each time the cell number reached 8 10 6 / ml, the cells were passaged, and the initial concentration was controlled at 1 10 6 / ml, after 60 passages, the dominant cell strain was enriched, and a stable high expression green fluorescent protein cell bank was obtained. The Top 1% cells were enriched by flow cytometry, and a stable high expression integrated dominant cell pool was obtained. After cell expansion, flow cytometry analysis was performed, and the fluorescence intensity was significantly increased. Figure 1 )。

[0063] Example 2, lentivirus integration site analysis:

[0064] The information of the stable integration site of the highly expressed exogenous protein was obtained by combining nest-PCR, next-generation sequencing technology, and bioinformatics technology.

[0065] First, the DNA of the above enriched Top 1% cells was extracted, and specific primers LTRI were designed for the 3' LTR region of the lentivirus vector. Linear PCR was performed using single-end DPO primers;

[0066] The PCR product was purified using AMPure XP magnetic beads. The VAHTS® ssDNA Library Prep Kit for Illumina kit was used to add adapters to the 3' end of the purified single-stranded DNA;

[0067] Extension Primer and Extension Enzyme Mix were used to complete double-stranded DNA synthesis, and double-stranded DNA was purified using AMPure XP magnetic beads. The purified DNA was used as a template, and Index PCR was performed using primers LTRII;

[0068] After the sequencing library was purified using AMPure XP magnetic beads, sequencing was performed;

[0069] Data analysis First, use python script to filter (LTR) reads containing specific exogenous sequences, with a maximum of 6 base mismatches, output the filtered fastq file; use cutadapt (version 2.8) tool to perform sequence truncation on the filtered fastq file, remove 66 bases (including primers and exogenous sequences) from the 5' end by parameter-u 66, get clean sequences containing only target insert fragments; use bwa mem (Version: 0.7.17-r1188) algorithm, with the specified complex reference genome (complexgenome.fna) as the template, set 4 threads (-t 4), align the processed sequence to the genome, and output the successfully aligned data to the genome as a sam format alignment result file; use awk command to extract the chromosome column and the aligned position column from the above sam file, the position is the insertion site, and then count the number of the same chromosome and the same position, and perform gene relative position annotation. In the range of 69195000-69199000 bases of CHO genome chromosome 6 (reference sequence NC_048599.1) (nucleotide sequence as shown in SEQ ID NO: 1), a total of 16791 reads were detected, which belongs to the intergenic position. According to the specific position, download the relevant reference sequence, design and construct the related expression vector, and perform exogenous protein stable high expression site verification.

[0070] Example 3, selection of target sequence:

[0071] According to the high expression exogenous protein stable site obtained by the above analysis, anti-PD1 antibody protein is selected to apply CRISPR / cas9 technology for site-specific integration. First, the target sequence of the site is designed. According to the selected site range (SEQ ID NO: 1), the sequence sgRNA is designed using DeepHF | SpCas9 & Base Editor Efficiency Prediction online prediction system, and the target sequence with higher efficiency is selected.

[0072] The online prediction system will score the editing efficiency of the recognized 5'NNNNNNNNNNNNNNNNNNNNNGG 3' target sequence. LOW efficacy (score<0.56); MEDIUM efficasy (0.56<=score<=0.74); HIGHefficacy (score>0.74).

[0073] The selected target sequence is as follows:

[0074] Target sequence 5'-TATCCCAAGTGAGACATGTGTGG-3' (SEQ ID No: 2), score = 0.689;

[0075] Target sequence 5'-ATGTGTGGATAATATAGTAATGG -3' (SEQ ID No: 3), score = 0.608;

[0076] Target sequence 5'-TCGTTTTATGGATAAAGCAATGG -3' (SEQ ID No: 4), score = 0.595;

[0077] Target sequence 5'-TCTATACCAGGTGTTTCAAGTGG-3' (SEQ ID No: 5), score = 0.584;

[0078] Target sequence 5'-AAACCCAGGACCATTGATTGAGG-3' (SEQ ID No: 6), score = 0.595;

[0079] Target sequence 5'-GCTTTCAGTTACAATTGTAGTGG-3' (SEQ ID No: 7), score = 0.587;

[0080] Target sequence 5'-AGCTTCTCTAAAAAAAGGACTGG-3' (SEQ ID No: 8), score = 0.593;

[0081] Target sequence 5'-AGCTACCTTCTGATGACAGAGGG-3' (SEQ ID No: 9), score = 0.607;

[0082] Target sequence 5'-AAATAGCCTATAGGGAGCAGAGG-3' (SEQ ID No: 10), score = 0.594;

[0083] Target sequence 5'-GTGGAATACTACATGCGCTTAGG-3' (SEQ ID No: 11), score = 0.604;

[0084] Target sequence 5'-GGTTGATACAGGGTTTCAGTGGG-3' (SEQ ID No: 12), score = 0.606;

[0085] Target sequence 5'-AGCAATTCTGTCACTAGTGAGGG-3' (SEQ ID No: 13), score = 0.603;

[0086] Target sequence 5'-CTCTGGTCCTTTGCAGATGAAGG-3' (SEQ ID No: 14), score = 0.604;

[0087] Target sequence 5'-ATTTAGGGAGATAAAGGTTGGGG-3' (SEQ ID No: 15), score = 0.606;

[0088] Target sequence 5'-GCTGAAAGGAGTTGAATGAAGGG-3' (SEQ ID No: 16), score = 0.594;

[0089] Further, the top three sequences with predicted editing efficiency were selected as target sequences, and the optimal target sequence SEQ ID No: 3 was selected according to the cleavage efficiency by the in vitro enzyme cleavage kit of Cas9.

[0090] Example 4, site-directed integration of anti-PD-1 protein gene with green fluorescent gene (GFP) and glutamine synthetase gene (GS)

[0091] The green fluorescent protein gene and the GS gene, and the anti-PD-1 protein gene were site-directed integrated at the target site by using the CRISPR / Cas9 mediated genome site-directed editing technology and homologous recombination. The CRISPR / Cas9 mediated homologous recombination technology needs to construct sgRNA plasmid and donor plasmid, and the construction process is as follows:

[0092] 1. Construction of sgRNA plasmid

[0093] 1) According to the target sequence selected in Example 3, the oligonucleotide chain was synthesized:

[0094] SgRNA-F: 5'-CACCGATGTGTGGATAATATAGTAA-3' (SEQ ID No: 17)

[0095] SgRNA-R: 5'-AAACTTACTATATTATCCACACATC-3' (SEQ ID No: 18).

[0096] 2) Annealing and ligation of the synthesized fragments

[0097] Reaction system: SgRNA-F (10 uM) 1 ul, SgRNA-R (10 uM) 1 ul, 10 x T4 ligase buffer 1 ul, ddH2O 7 ul, total volume 10 ul.

[0098] PCR instrument 95 °C for 5 min, 4 °C storage.

[0099] 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).

[0100] 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.

[0101] The reaction procedure is as follows:

[0102] 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.

[0103] 4) Conversion to DH5 Sensitive state;

[0104] 5) Select positive single clones and sequence them using the universal primer sgRNA-CX-F:5'-GCCTATTTCCCATGATTCCTTC-3' (SEQ ID No:19);

[0105] 6) Expand positive monoclonal strains and extract plasmids.

[0106] 2. Donor plasmid construction

[0107] Specific information about the Donor plasmid is as follows: Figure 4 As shown, this plasmid mainly includes the anti-PD1 protein expression gene, the GS selection gene, GFP and mcherry positive and negative selection genes, two homologous recombination arms (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:

[0108] 1) The 5' homologous arm fragment and the 3' homologous arm fragment were derived from the genomic DNA template, and the mChery 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.

[0109] 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.

[0110] 3) Select positive monoclonal, sequencing, expansion and extraction of plasmid.

[0111] 3. Screening and identification of single copy site-specific integration of NC_048599.1 at base 69197141

[0112] The constructed sgRNA plasmid ( Figure 3 ), donor plasmid ( Figure 4 ), and Cas9 plasmid containing Cas9 protein ( Figure 2 ) were co-transfected into CHO-S-KOGS cells cultured at 37°C and 5% CO2. The molar ratio of the three plasmids was 2:1:1. The cells were transfected using an electroporator and screened using a glutamine-free medium. The 5' arm and 3' arm of the donor plasmid contain the N20+PAM sequence of the target gene, so after co-transfection of the three plasmids, the donor plasmid will be recognized and cut into a linear fragment to improve integration efficiency. Through homologous recombination, the target gene is integrated at the target site under the selection pressure of the glutamine-free medium. At the same time, mcherry in the donor plasmid serves as a negative selection marker, which can exclude random integration of monoclonal cells during screening and reduce the workload of screening site-specific integration of monoclonal cell lines. When the cell viability is stable and the proportion of green fluorescence increases, use a BD flow cytometer to sort monoclonal cells, select cells that express green fluorescence but not red fluorescence, and inoculate them into 96-well plates.

[0113] After 14 days of monoclonal culture, collect the culture supernatant of cells expressing green fluorescence but not red fluorescence, take 3 μL and mark it on the NC membrane, dry it at 37°C, block it with 5% skim milk for 30 minutes, wash it with TBST three times, incubate it with goat anti-rabbit secondary antibody (1 μg / mL) for 1 hour, wash it with TBST three times, and then use ECL to develop and detect the cell strain expressing and secreting anti-PD1 protein. According to the spot hybridization results of the monoclonal supernatant, 24 positive monoclonal strains are preliminarily screened out ( Figure 7 );

[0114] Positive monoclonal cells were expanded, and a portion of the genome was extracted for PCR identification. Through 5' junction PCR, 3' junction PCR, Sanger sequencing analysis ( Figure 8 ), and copy number identification, 12 monoclonal cell strains with single copy integration of anti-PD1 gene were screened out ( Figure 9 ). The above obtained monoclonal cell strains were expanded.

[0115] Test example:

[0116] Octet molecular interaction instrument was used to detect the anti-PD1 protein expression of the monoclonal cell strains obtained in Example 4:

[0117] The 12 monoclonal cell strains obtained in Example 4 were subjected to plate fermentation, inoculated at an initial concentration of 2 10 5 / ml of cells, and the supernatant was collected after 6 days of fermentation. The anti-PD1 protein expression in the fermentation broth was detected using Protein A sensor by Octet molecular interaction instrument. The results showed that the single copy integrated protein expression was between 0.91-11.9 μg / ml Figure 10 , with an average of 5.2 μg / ml.

[0118] Four of the monoclonal M4-10, M4-14, M4-17, and M57-22 were selected for continuous 20-week plate fermentation, and the protein expression stability was detected at weeks 4, 8, 12, 16, and 20. The results showed that the monoclonal cell strains could stably express anti-PD1, indicating that the site had good expression stability, as shown in Figure 11 .

[0119] Three of the monoclonal cell strains were selected for every-other-day fed-batch fermentation. The cells were inoculated at a concentration of 6 10 5 / ml on day 0, and the feeding was started on day 4. The EmCD CHO101 feed A medium was added at a volume of 6%, and the EmCD CHO101 feed B medium was added at a volume of 0.6%. The glucose was maintained at 4 g / L. The cells were collected when the cell viability was 80%, and the anti-PD1 protein expression in the fermentation broth was detected using Protein A sensor. The results showed that the protein expression of the single copy integrated anti-PD1 gene in the fed-batch fermentation was between 24.4-70.4 μg / ml, with an average of 45.6 μg / ml, as shown in Figure 12 .

[0120] In the embodiments of the present application, CRISPR / Cas9-mediated genome site-directed editing technology is mainly used for site-directed integration. Therefore, the target sequences are mainly designed for the upstream and downstream sequences within 4000 bp of bases. The 15 groups of target sequences screened in Example 3 of the present application cover most of the upstream and downstream sequences within 4000 bp of bases in the present application. The range of 69195000-69199000 bases in the CHO cell gene NC_048599.1 can successfully construct a site-directed integration stable expression cell line, and can stably express the target protein.

[0121] The above-described embodiments are merely preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent substitutions or transformations made by those skilled in the art based on the present application are within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. The application of a site for stable expression of a target protein in CHO cells in the stable expression of the target protein, characterized in that, The site for stable expression of the target protein is located on chromosome 6 of CHO cells within the nucleotide sequence shown in SEQ ID NO:1, and the application involves inserting an exogenous target protein gene at this site.

2. The application as described in claim 1, characterized in that, The site where the target protein is stably expressed is located on chromosome 6 in CHO cells, such as in the range of bases 69196500-69197500 of the reference sequence NC_048599.

1.

3. The application as described in claim 2, characterized in that, Within the base range of 69196500-69197500, the target sequence that can be recognized by CRISPR / Cas9 technology is 5'-ATGTGTGGATAATATAGTAATGG-3'.

4. The application as described in claim 1, characterized in that, The target protein is a protein with a molecular weight of less than 160 kDa.

5. The application as described in claim 4, characterized in that, The target protein is a functional protein.

6. The application as described in claim 4, characterized in that, The target protein is a polypeptide.

7. The application as described in claim 4, characterized in that, The target protein is a fusion protein.

8. An expression vector for expressing a target protein in CHO cells, characterized in that, It is an expression vector used to insert the gene encoding the target protein into a stable expression site of the target protein in CHO cells; The site where the target protein is stably expressed is located on chromosome 6 in CHO cells, such as in the range of bases 69196500-69197500 of the reference sequence NC_048599.

1.

9. The expression vector as described in claim 8, characterized in that, These include site-specific integration vectors based on recombinases, site-directed insertion vectors for gene editing, or integration vectors based on homology-directed repair.

10. The expression vector as described in claim 8, characterized in that, 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. The 5' and 3' homologous arms are the upstream and downstream homologous arms of 5'-ATGTGTGGATAATATAGTAATGG-3' on chromosome 6, which can be recognized by CRISPR / Cas9 technology.

11. The expression vector as described in claim 8, characterized in that, The expression vector further includes a promoter sequence located upstream of the coding gene of the target protein, wherein the promoter controls the expression of the protein; The promoters are selected from: CMV, EF-1a, SV40 and the combined promoter CAG.

12. A CHO recombinant cell line, characterized in that, It includes inserting an exogenous target protein gene at a site where the target protein is stably expressed. The site where the target protein is stably expressed is chromosome 6 in CHO cells, such as the nucleotide sequence of reference sequence NC_048599.1, within the range of bases 69196500-69197500.

13. The CHO recombinant cell line as described in claim 12, characterized in that, It is obtained by integrating the target protein gene into chromosome 6 of CHO cells using the expression vector as described in any one of claims 8 to 11, with the integration site located in the range of bases 69196500-69197500 of the reference sequence NC_048599.1.

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