Glutamine synthetase gene knockout Chinese hamster ovary cell strain and application thereof

Abstract

The invention relates to the field of bioengineering, and discloses a Chinese hamster ovary cell strain with a glutamine synthetase gene knocked out and application of the Chinese hamster ovary cell strain. The Chinese hamster ovary cell strain disclosed by the invention is a monoclonal cell strain which is obtained by carrying out glutamine synthetase (GS) gene knockout on a CHO-K1 cell strain and then carrying out subcloning screening. In the GS gene knockout process of the Chinese hamster ovary cell strain, no resistance gene is introduced, no Cas9 gene participates, no exogenous sequence is integrated into a cell genome, and the safety is high. Compared with a common GS gene knockout CHO-K1 cell strain, the GS gene knockout CHO-K1 cell strain has the characteristics of good growth, short target recombinant protein expression screening period and high expression quantity.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a glutamine synthetase gene knocked-out Chinese hamster ovary cell strain and application thereof. Background Art

[0002] Chinese hamster ovary cells (CHO cel1) are a continuous cell line that can be propagated for more than a hundred generations and are immortal. They are currently the most widely recognized cell line in bioengineering. CHO cells have many significant characteristics, such as the ability to produce post-translationally modified proteins similar to those of humans and the ability to undergo complex glycosylation; CHO cells are non-secreting cells that rarely secrete CHO endogenous proteins themselves, and CHO cells are also easy to transfect, making them a common choice for transient and stable expression in research; CHO cells have good genomic background characteristics and have a relatively fast growth rate and high protein expression yield in suspension culture; in addition, CHO cells proliferate rapidly, making them very suitable for large-scale culture, and have the advantages of high stability and easy operation. CHO cell large-scale culture technology and its bioreactor engineering can be widely used in the research, development and industrial production of biotechnology products such as antibodies, recombinant protein drugs, and viral vaccines.

[0003] After more than 60 years of research history, CHO cells have derived a variety of cell lines with different properties from the original cell line. CHO-K1 cells are one of them and are widely used. There are two screening systems in CHO cells: dihydrofolate reductase (DHFR) and glutamine synthetase (GS). The protein screening system mainly used in the CHO-K1-based cell line is GS (glutamine synthetase) and the antibiotic screening system has also improved the screening efficiency. Currently, most of the therapeutic proteins that have been launched on the market are developed and produced based on CHO-K1 cells. The Horizon Discovery CHO engineered cell line is derived from ECACC (European Cell Bank) CHO-K1 cells. Using Horizon Discovery's recombinant adenovirus (rAAV) technology, the sixth exon (an essential element for enzyme catalytic activity) of the GS gene (glutamine synthetase) is knocked out through homologous recombination to obtain a CHO-K1 cell line without the glutamine synthetase gene. The cell line is further domesticated into suspension cells and adapted to the existing commercial chemical-defined animal-free culture medium, thereby obtaining a CHO cell line that stably produces protein drugs / antibody drugs - HD-BioP3.

[0004] The GS screening system is a newly developed and more efficient amplification expression screening system. The culture and growth process of CHO cells requires glutamine (L-glutamine). GS converts glutamate and ammonia into glutamine, and animal cells obtain glutamine through this enzymatic reaction. In the GS screening system, the recombinant protein expression vector carrying exogenous GS is first transfected into CHO cells. CHO cells obtain exogenous GS and express it, and use glutamate and ammonia in the culture medium to synthesize glutamine for cell culture and growth. Therefore, the culture medium used before and during transfection should be a complete culture medium with glutamine added. When the plasmid is transfected and integrated into CHO cells, the culture medium without glutamine is used so that only cells transfected with the exogenous GS gene and the target protein gene can grow, achieving the purpose of screening and obtaining recombinant expression cell lines. During the culture process, if the GS inhibitor methionine sulfhydrylamine (MSX) is added, the expression of the GS gene can be inhibited. Only when the GS gene is amplified to a large number of copies can it survive. Therefore, under the drug pressure of MSX, the anti-MSX cell line obtained is a high-level expression of the GS gene, and also a high-level expression of the target gene, thereby obtaining a recombinant expression screening strain with higher expression. On this basis, CHO-K1 (GS- / -) cells with GS gene knocked out completely lack endogenously expressed GS. When cultured without glutamine and screened with MSX, a recombinant expression screening strain with higher expression than cells with endogenous GS can be obtained. Therefore, GS knocked out defective CHO-K1 cells are widely used in the fields of biopharmaceuticals and biological raw material production.

[0005] However, in the process of GS gene knockout in CHO cells, some exogenous genes such as resistance genes and Cas9 genes are usually introduced into the cells, which not only affects the safety, but also causes the Chinese hamster ovary cell line obtained after gene knockout to have a long screening cycle and low expression level of the target recombinant protein expression during application. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a Chinese hamster ovary cell line with glutamine synthetase gene knockout and its application. The Chinese hamster ovary cell line of the present invention is a monoclonal cell line obtained by subclone screening after glutamine synthetase (GS) gene knockout of CHO-K1 cell line. In the process of GS gene knockout of the Chinese hamster ovary cell line of the present invention, no resistance gene is introduced, no Cas9 gene is involved, no exogenous sequence is integrated into the cell genome, and the safety is high. Compared with the CHO-K1 cell line with ordinary GS gene knockout, it has the characteristics of good growth, short screening cycle of target recombinant protein expression and high expression level.

[0007] The specific technical scheme of the present invention is: In a first aspect, the present invention provides two Chinese hamster ovary cell lines with glutamine synthetase gene knockout, wherein: The first strain was named Chinese Hamster Ovary Cell CHO-K1 / LG-GSKa1, and the microbial classification was named ChineseHamster Ovary. It was deposited in the China Center for Type Culture Collection on May 29, 2024; the deposit number is CCTCCNO: C2024149.

[0008] The second strain was named Chinese Hamster Ovary Cell CHO-K1 / LG-GSKb2, and the microbial classification was named Chinese Hamster Ovary. It was deposited in the China Center for Type Culture Collection on May 29, 2024; the preservation number is CCTCCNO: C2024167.

[0009] The two Chinese hamster ovary cell lines of the present invention are monoclonal cell lines obtained by subclone screening after knocking out the glutamine synthetase (GS) gene of the CHO-K1 cell line. In the process of knocking out the GS gene, no resistance gene is introduced into the two Chinese hamster ovary cell lines of the present invention, no Cas9 gene is involved, and no exogenous sequence is integrated into the cell genome, so the safety is high. Compared with the CHO-K1 cell line with ordinary GS gene knockout, it has the characteristics of good growth, short screening cycle of target recombinant protein expression and high expression level.

[0010] Specifically, the Chinese hamster ovary cell CHO-K1 / LG-GSKa1 is a subclone cell strain of the CHO-K1 cell line with double knockout of the glutamine synthetase (GS) gene on chromosome 5 and chromosome 1. The Chinese hamster ovary cell CHO-K1 / LG-GSKb2 is a subclone cell strain of the CHO-K1 cell line with double knockout of the glutamine synthetase (GS) gene on chromosome 5.

[0011] More specifically, the gene sequence knocked out on chromosome 5 is a 5385 bp sequence from intron 1 to exon 7 of the glutamine synthetase gene, as shown in SEQ ID NO.35; the gene sequence knocked out on chromosome 1 is a 1325 bp sequence of the glutamine synthetase gene including the entire exon, as shown in SEQ ID NO.36.

[0012] In a second aspect, the present invention provides the use of the above-mentioned Chinese hamster ovary cell line in the expression of recombinant proteins.

[0013] In a third aspect, the present invention provides a method for obtaining the above-mentioned Chinese hamster ovary cell line, comprising: 1) Analyze the glutamine synthetase gene on chromosomes 5 and 1 of CHO-K1 cells, develop a knockout plan, and design the Cas9-guided sgRNA sequence that recognizes the glutamine synthetase gene; 2) Connect the tracrRNA sequence after the sgRNA sequence and synthesize the PCR reaction primers; 3) PCR amplification to obtain the sgRNA in vitro transcription template; 4) Obtaining sgRNA containing sgRNA sequence and tracrRNA sequence by in vitro transcription with RNA polymerase; 5) The sgRNA is combined with the Cas9 protein to form an RNP complex, which is then transfected into CHO-K1 cells; 6) Monoclonal culture to obtain a CHO-K1 monoclonal cell line with glutamine synthetase gene knockout.

[0014] 7) Expand the culture and cryopreservation of the CHO-K1 monoclonal cell line with glutamine synthetase gene knockout; 8) The glutamine synthetase gene knockout CHO-K1 monoclonal cell line obtained in 7) was verified for RNA level, protein level and functional defect of glutamine synthetase, and after screening, a high-expression glutamine synthetase gene knockout CHO-K1 subclone cell line was obtained.

[0015] In the prior art, when performing gene editing in CHO cells, the method of introducing Cas9 and sgRNA is usually to construct a plasmid vector carrying Cas9 and sgRNA, and then electro-transfect the plasmid vector into the cell. This delivery method has the following disadvantages: the plasmid vector can exist in the cell for a long time and continuously express Cas9 and sgRNA; the plasmid vector is difficult to transfect because it carries Cas9 and sgRNA; the preparation of the carrier vector of Cas9 and sgRNA is time-consuming and costly; the plasmid vector will introduce resistance genes and screening genes that are not needed later.

[0016] The method of the present invention is used to deliver Cas9 and sgRNA into CHO cells. This method does not introduce any vectors containing resistance genes and screening genes into the cells, which can avoid damage to CHO cells. The RNP complex will be degraded and there is no possibility of being integrated into the host genome. Finally, because the RNP complex does not need to undergo a translation process in the cell to obtain the Cas9 protein, it can start to work once it is delivered into the cell, so a higher cutting efficiency can be obtained. In addition, the method of the present invention can avoid the construction of expensive and time-consuming plasmid vectors, and can shorten the cycle of GS knockout.

[0017] Compared with the prior art, the beneficial effects of the present invention are: (1) The Chinese hamster ovary cell line of the present invention is a monoclonal cell line obtained by subclone screening after knocking out the glutamine synthetase (GS) gene of the CHO-K1 cell line. In the process of knocking out the GS gene, the Chinese hamster ovary cell line of the present invention does not introduce any resistance gene, does not involve the Cas9 gene, and does not integrate exogenous sequences into the cell genome, so it has high safety. Compared with the ordinary GS gene knocked out CHO-K1 cell line, it has the characteristics of good growth, short target recombinant protein expression screening cycle and high expression level.

[0018] (2) In the process of delivering Cas9 and sgRNA into CHO cells using the method of the present invention, no vector containing resistance genes and screening genes is introduced into the cells, which can avoid damage to CHO cells. The introduced RNP complex will be degraded and there is no possibility of being integrated into the host genome. In addition, the RNP complex does not need to undergo the process of translation to obtain Cas9 in the cell, and it can start to work once it is delivered into the cell, so the cutting efficiency is higher. The method of the present invention can avoid the construction of expensive and time-consuming plasmid vectors and shorten the cycle of GS knockout. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Construction of a strategy map for the knockout of GS genes.

[0020] Figure 2 It is the guide RNA for GS gene targeted knockout obtained by in vitro transcription.

[0021] Figure 3 This is a DNA agarose gel electrophoresis verification diagram of GS gene knockout.

[0022] Figure 4 This is the Sanger sequencing verification diagram of GS gene knockout.

[0023] Figure 5 The changes in GS RNA levels in GS gene knockout cells.

[0024] Figure 6 Verification of GS functional deficiency in GS gene knockout cells.

[0025] Figure 7 This is the recombinant expression cycle of the GS gene knockout monoclonal cell line.

[0026] Figure 8 is the expression level of GS gene knockout monoclonal cell line. DETAILED DESCRIPTION

[0027] In order to illustrate the technical means and effects of the present invention, the present invention is further described in detail below with reference to examples and drawings. However, the embodiments of the present invention include but are not limited to these.

[0028] Overall embodiment First, the present invention provides two Chinese hamster ovary cell lines with glutamine synthetase gene knockout, wherein: The first strain was named Chinese Hamster Ovary Cell CHO-K1 / LG-GSKa1, and the microbial classification was named ChineseHamster Ovary. It was deposited in the China Center for Type Culture Collection on May 29, 2024; the deposit number is CCTCCNO: C2024149.

[0029] The second strain was named Chinese Hamster Ovary Cell CHO-K1 / LG-GSKb2, and the microbial classification was named Chinese Hamster Ovary. It was deposited in the China Center for Type Culture Collection on May 29, 2024; the preservation number is CCTCCNO: C2024167.

[0030] Specifically, the Chinese hamster ovary cell CHO-K1 / LG-GSKa1 is a subclone cell strain of the CHO-K1 cell line with double knockout of the glutamine synthetase (GS) gene on chromosome 5 and chromosome 1. The Chinese hamster ovary cell CHO-K1 / LG-GSKb2 is a subclone cell strain of the CHO-K1 cell line with double knockout of the glutamine synthetase (GS) gene on chromosome 5.

[0031] More specifically, the gene sequence knocked out on chromosome 5 is a 5385 bp sequence from intron 1 to exon 7 of the glutamine synthetase gene, as shown in SEQ ID NO.35; the gene sequence knocked out on chromosome 1 is a 1325 bp sequence of the glutamine synthetase gene including the entire exon, as shown in SEQ ID NO.36.

[0032] Secondly, the present invention provides the use of the Chinese hamster ovary cell line in the expression of recombinant proteins.

[0033] Finally, the present invention provides a method for preparing a GS gene knockout CHO-K1 monoclonal cell line, comprising the following steps: 1) Analyze the GS gene on chromosome 5 and chromosome 1 of CHO-K1 cells, develop a knockout plan and design the Cas9-guided sgRNA sequence (i.e., crRNA) that recognizes the GS gene.

[0034] In some embodiments, two sgRNAs are designed for the GS gene on each chromosome; wherein: for chromosome 5, there are sgRNA sequences targeting intron 1 and sgRNA sequences targeting exon 7; for chromosome 1, there are sgRNA sequences targeting the 5' end and sgRNA sequences targeting the 3' end. Further preferably, the sgRNA sequence targeting intron 2 is shown in SEQ ID NO.17; the sgRNA sequence targeting exon 7 is shown in SEQ ID NO.18; the 5' end targeting sgRNA sequence is shown in SEQ ID NO.19; and the 3' end targeting sgRNA sequence is shown in SEQ ID NO.20.

[0035] 2) Connect the tracrRNA sequence to the sgRNA sequence and synthesize the PCR reaction primers.

[0036] In some embodiments, the tracrRNA sequence is as shown in SEQ ID NO. 21. The PCR reaction primers include: for chromosome 5: the forward primer sequence of the sgRNA targeting intron 1 is shown in SEQ ID NO. 22; the forward primer sequence of the sgRNA targeting exon 7 is shown in SEQ ID NO. 23; for chromosome 1: the forward primer sequence of the 5' end targeting sgRNA is shown in SEQ ID NO. 24; the forward primer sequence of the 3' end targeting sgRNA is shown in SEQ ID NO. 25.

[0037] 3) Perform PCR amplification using PCR reaction primers, recover and purify to obtain a large amount of sgRNA in vitro transcription templates.

[0038] In some embodiments, the recovery concentration of the sgRNA in vitro transcribed template is 0.5-1.5 μg / μl, and its purification method includes but is not limited to gel recovery and phenol chloroform extraction.

[0039] 4) Using the sgRNA in vitro transcription template, in vitro transcription under the action of RNA polymerase obtains an sgRNA containing the sgRNA sequence and the tracrRNA sequence.

[0040] In some embodiments, the RNA polymerase is T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase.

[0041] 5) The sgRNA is combined with the Cas9 protein to form an RNP complex, which is then transfected into CHO-K1 cells.

[0042] In some embodiments, the transfection method is electrofection or liposome transfection. The source of the CHO-K1 cells is ATCC: CCL-61.

[0043] 6) The transfected CHO-K1 cells were subjected to monoclonal culture to obtain a CHO-K1 monoclonal cell line with GS biallelic gene knockout.

[0044] In some embodiments, the monoclonal culture method is: statically culture the transfected CHO-K1 cells, pick up round, medium-sized cells under a microscope using a mouth pipette, place one cell in each well, perform monoclonal plating, and check at two time points, 2 hours later and within 24 hours, to determine the monoclonal cell wells and exclude non-monoclonal cell wells.

[0045] In some more specific embodiments, the static culture medium is OPM-Altair CHO medium.

[0046] In some more specific embodiments, the monoclonal plating methods include but are not limited to mouth pipette selection, limiting dilution method and using Andawang VIPS PRO plating instrument.

[0047] In some more specific embodiments, when the confluence rate of monoclonal cells is 55-65%, a portion of the cells are taken for DNA extraction, PCR detection and Sanger sequencing verification to obtain a CHO-K1 monoclonal cell line with GS biallelic gene knockout.

[0048] In some more specific embodiments, the method of DNA extraction includes but is not limited to alkaline lysis and phenol-chloroform extraction.

[0049] 7) The CHO-K1 monoclonal cell line with GS double allele knockout was expanded and cryopreserved.

[0050] In some embodiments, the expanded culture is subcultured for more than 3 generations.

[0051] 8) The GS biallelic knockout CHO-K1 monoclonal cell line obtained in 7) was subjected to GS RNA level, protein level and functional defect verification (including fluorescence quantitative PCR, Western Blot and cell growth curve observation, etc.), and after screening, a high-expression GS biallelic knockout CHO-K1 monoclonal cell line was obtained.

[0052] In some embodiments, 8) specifically includes: A) A CHO-K1 monoclonal cell line with biallelic knockout of GS was subcloned and plated by mouth pipette selection.

[0053] In some more specific embodiments, in A), the plating method includes but is not limited to the mouth pipette selection method, the limiting dilution method and the Andawang VIPS PRO plating instrument.

[0054] B) The subclone cells that first grow to 70-80% confluence and have uniform cell size are expanded and cryopreserved.

[0055] C) Transfecting the constructed recombinant protein expression vector into the subclone cell line obtained in B).

[0056] In some more specific embodiments, the transfection method includes but is not limited to electro-transfection. The recombinant protein expression vector is a vector that has been constructed in advance and verified to be able to express normally.

[0057] D) The transfected subcloned cell lines were placed in medium containing glutamine to recover.

[0058] In some more specific embodiments, the recovery culture method is static culture in a T25 flask, and the glutamine concentration is 3-5 mM.

[0059] E) The recovered subcloned cell lines were placed in a medium without glutamine for screening.

[0060] F) Continuously monitor the density and viability of subcloned cell lines.

[0061] G) When the cell viability reaches above 90%, the growth rate is normal and can be stable for 3 generations, single clones are plated by mouth pipette selection method.

[0062] H) When the growth confluence rate of the monoclonal cell line in G) reaches 45-55%, ELISA reaction screening is performed to select the monoclonal cell line with high expression level.

[0063] I) The monoclonal clone obtained in H) is expanded and cultured, and Fed-Batch induced expression is performed.

[0064] In some more specific embodiments, in I), the induced expression time is 12-16 days.

[0065] J) Monitor the expression level of the cell lines in I), analyze the time required for each monoclonal cell line from E) to G), and analyze the expression level of the recombinant protein to select the optimal subclone cell line. Specific embodiments Example 1: GS gene knockout The GS gene was found on chromosome 5 and consists of 7 exons. The CDS region starts from the second exon. Exon 6 was found to be an important component of the GS gene. A base sequence on it is the location of the active site. Its sequence is shown in SEQ ID NO.10 (the exon 6 sequence is underlined, and the active site sequence is double underlined). Marker); on chromosome 1, there is a pseudogene of the GS gene, which consists of one exon. The sequence on this exon is highly similar to the CDS sequence of the GS gene on chromosome 5, including the action site sequence on exon 6, and its sequence is shown in SEQ ID NO.15 (the exon 6 sequence is underlined). __ Mark, the action site sequence uses double underscores Based on this, the knockout strategy of GS gene is as follows Figure 1 As shown, for the GS gene on CHO-K1 chromosome 5, two sgRNAs were designed to target intron 1 and exon 7 respectively, and the sequence between the two was deleted; for the GS gene on CHO-K1 chromosome 1, two sgRNAs, 5' and 3', were designed to target and knock out its entire exon.

[0067] The sequence of intron 1 of the GS gene of chromosome 5 is shown as SEQ ID NO. 1; The sequence of exon 2 of the GS gene on chromosome 5 is shown in SEQ ID NO. 2; The sequence of intron 2 of the GS gene of chromosome 5 is shown in SEQ ID NO. 3; The sequence of exon 3 of the GS gene on chromosome 5 is shown in SEQ ID NO. 4; The sequence of intron 3 of the GS gene of chromosome 5 is shown as SEQ ID NO. 5; The sequence of exon 4 of the GS gene on chromosome 5 is shown in SEQ ID NO. 6; The sequence of intron 4 of the GS gene of chromosome 5 is shown in SEQ ID NO. 7; The sequence of exon 5 of the GS gene on chromosome 5 is shown in SEQ ID NO. 8; The sequence of intron 5 of the GS gene of chromosome 5 is shown in SEQ ID NO.9; The sequence of exon 6 of the GS gene on chromosome 5 is shown in SEQ ID NO.10:

[0068] The sequence of intron 6 of the GS gene of chromosome 5 is shown as SEQ ID NO. 11; The sequence of exon 7 of the GS gene on chromosome 5 is shown in SEQ ID NO.12: The sequence of the 3'UTR of the GS gene on chromosome 5 is shown as SEQ ID NO. 13; The sequence of the 5'UTR of the GS gene on chromosome 1 is shown in SEQ ID NO. 14; The sequence of the exon of the GS gene on chromosome 1 is shown in SEQ ID NO.15: The sequence of the 3'UTR of the GS gene on chromosome 1 is shown as SEQ ID NO.16.

[0069] (1) Comprehensively analyze the exon 2 to exon 7 region of the GS gene on chromosome 5 and design the targeting sgRNA sequence, the sequence of which is as follows: sgRNA sequence targeting intron 1: SEQ ID NO.17; sgRNA sequence targeting exon 7: SEQ ID NO.18.

[0070] (2) The exon sequences of the GS gene on chromosome 1 were comprehensively analyzed, and the targeting sgRNA sequences were designed as follows: 5′ end targeting sgRNA sequence: SEQ ID NO.19; 3' end targeting sgRNA sequence: SEQ ID NO.20.

[0071] (3) Synthesize sgRNA primers (Suzhou Jinweizhi), use sgRNA primer pairs to perform PCR reaction, and obtain a large number of sgRNA in vitro transcription templates. The template used in the PCR reaction is pUC-sgRNA containing tracrRNA, and the system is: ddH2O 6μL, sgRNA F 0.5μL, sgRNA R 0.5μL, template 1μL, 2xTaq master mix (CW0682) 7.5μL. The program is: 95℃ 3 minutes, 95℃ 30 seconds, 64℃ 30 seconds, 72℃ 10 seconds, and 34 cycles.

[0072] The tracrRNA sequence is shown in SEQ ID NO.21; The forward primer sequence of the sgRNA targeting intron 1 is shown in SEQ ID NO. 22; The forward primer sequence of the sgRNA targeting exon 7 is shown in SEQ ID NO.23: The forward primer sequence targeting sgRNA at the 5' end is shown in SEQ ID NO.24; The forward primer sequence targeting sgRNA at the 3' end is shown in SEQ ID NO.25.

[0073] (4) The PCR product obtained in step (3) was excised and recovered (DP209-02) to a concentration of 1 μg / μL.

[0074] (5) In vitro transcription of sgRNA (Invitrogen, AM1333), reaction system: ATP 2 μL, GTP 2 μL, CTP 2 μL, UTP 2 μL, 10x buffer 2 μL, T7 Polymerase 2 μL, sgRNA template (purified in step (3)) 1 μL, ddH20 7 μL; reaction conditions: incubation at 37°C for 2-4 hours.

[0075] (6) Take 1 μL of the product from step 4 and perform electrophoresis on a 2% agarose gel for verification (see Figure 2 ), successfully obtaining high-quality sgRNA.

[0076] (7) Add 1 μL of DNaseI to the sgRNA in vitro transcription reaction system in step (5) and digest at 37°C for 15 minutes, then add EDTA to terminate the reaction, followed by chloroform extraction and ethanol precipitation to recover and purify the sgRNA.

[0077] (8) Discard the ethanol in step (7), place in a vacuum concentrator to dry, then add 80 μL of Nuclease-free water to dissolve the sgRNA, and store at -80°C for later use.

[0078] (9) Prepare the RNP complex on the day of electroporation, and combine the Cas9 protein with the sgRNA at room temperature to obtain the RNP complex.

[0079] (10) Prepare CHO-K1 (ATCC: CCL-61) suspension cells in advance, resuscitate and stabilize for 3 generations, and then take cells in the logarithmic growth phase on the day of electroporation for electroporation using the Neon transfection system. The electroporation conditions are: cell density 2x10 7 cells / mL, 100μL system (Thermo fisher, MPK10025), 1600V / 20msec / 1pulse.

[0080] (11) The electroporated CHO-K1 cells were allowed to recover for 48 h in OPM-Altair medium (4 mM glutamine) and single colonies were plated using the mouth pipette selection method.

[0081] (12) When the confluence rate of the single clone reaches about 60%, remove some cells and centrifuge at 12000g for 2 minutes. Add 100 μL of 50 mM NaOH and heat in a metal bath at 95°C for 10 minutes. Remove and add 100 μL of TE, vortex and centrifuge at 12000g for 2 minutes.

[0082] (13) Take 1 μL of the supernatant in step (12) and perform PCR reaction to verify the knockout of GS. A total of 6 pairs of primers were designed for the knockout of GS. When naming the primers below, the GS gene on chromosome 5 is referred to as GS5, and the GS gene on chromosome 1 is referred to as GS1. The primer pair sequences are as follows: GS5-WT5'F: SEQ ID NO.26; GS5-WT5'F1: SEQ ID NO.27; GS5-WT5'R: SEQ ID NO.28; GS5-WT3'F: SEQ ID NO.29; GS5-WT3'R: SEQ ID NO.30; GS1-WT5'F: SEQ ID NO.31; GS1-WT5'R: SEQ ID NO.32; GS1-WT3'F: SEQ ID NO.33; GS1-WT3'R: SEQ ID NO.34.

[0083] (14) In step (13), the primer pair of GS5-WT5'F1 (SEQ ID NO.27) and GS5-WT3'R (SEQ ID NO.30) and GS1-WT5'F (SEQ ID NO.31) and GS1-WT3'R (SEQ ID NO.34) are primers for identifying knockout cells, and their PCR products contain sequences at both ends of the knockout fragment. In the knockout cell line, the product is short due to the lack of the knockout sequence and can be detected, while in the non-knockout cell line, the product is as long as about 7kb and cannot be amplified. See Figure 3 As shown in the figure, lane numbered 1 is cell line B, and lane numbered 20 is cell line A. The products of the above two primer pairs were excised and recovered, and Sanger sequencing was performed for verification.

[0084] Experimental results: Sanger sequencing results are shown in Figure 4 As shown, the sequence targeted by sgRNA on the GS gene on chromosome 5 was successfully knocked out, including its intron 2 to exon 7 (SEQ ID NO.35); the sequence of the GS gene on chromosome 1 including the entire exon (SEQ ID NO.36) was successfully knocked out, indicating that the GS knockout cell line was successfully obtained. Among them, the GS5 and GS1 double knockout cell line A (GS5- / -; GS1- / -) and the GS5 double knockout cell line B (GS5- / -) were screened.

[0085] SEQ ID NO.35: GS5 knockout sequence, with a total length of 5385 bp, including sequences from intron 1 to exon 7; SEQ ID NO.36: GS1 knockout sequence, with a total length of 1325 bp, including the entire exon sequence.

[0086] Example 2: GS knockout validation (1) RT-PCR primers were designed for the GS gene to detect the loss of GS at the RNA level. The primer pair sequences are as follows: GS5-E1F: SEQ ID NO.37; GS5-E2R: SEQ ID NO. 38; GS5-E2F: SEQ ID NO. 39; GS5-E3R: SEQ ID NO.40; GS5-E6F: SEQ ID NO.41; GS5-E7R: SEQ ID NO.42; GS1-F: SEQ ID NO.43; GS1-R: SEQ ID NO.44.

[0087] (2) Take 1x10 6 The wild-type CHO-K1 cells, cell line A cells and cell line B cells were centrifuged at 12000g for 2 minutes and the supernatant was discarded.

[0088] (3) According to the operating manual of QIAGENE (74104) RNeasyminikit, add 350 μL of cell lysis buffer to each of the three groups of cells in step (2), grind with a small electric grinder for 15 seconds, and immediately add 350 μL of 70% anhydrous ethanol, and mix by gently pipetting up and down with a pipette.

[0089] (4) Add the supernatant from step (3) into a 2 mL centrifuge tube containing an adsorption column, centrifuge at 8000 g for 30 seconds, and discard the liquid.

[0090] (5) Add 700 μL of liquid RW1 to the adsorption column, centrifuge at 8000 g for 30 seconds, and discard the liquid.

[0091] (6) Add 500 μL of liquid RPE to the adsorption column, centrifuge at 8000 g for 30 seconds, and discard the liquid.

[0092] (7) Add 500 μL of liquid RPE to the adsorption column, centrifuge at 8000 g for 2 minutes, and discard the liquid.

[0093] (8) Discard the liquid and place the adsorption column back into a 2 mL centrifuge tube. Centrifuge at 12,000 g for 2 minutes.

[0094] (9) Remove the adsorption column and place it in a new 1.5 mL centrifuge tube. Add 40 μL of nuclease-free water and centrifuge at 12,000 g for 2 minutes. Use Nanodrop to measure RNA concentration.

[0095] (10) Take equal amounts of RNA (500-1 μg) from the three groups of cells obtained in step (9) and transcribe using the Bio-Rad iScript cDNA synthesis kit. The reaction system is: 4 μL of 5x iScript reaction solution, 1 μL of iScript reverse transcriptase, RNA (500-1 μg), and nuclease-free water to 20 μL. The reaction conditions are: 25°C for 5 minutes, 46°C for 20 minutes, 95°C for 1 minute, and storage at 4°C.

[0096] (11) Take 1 μL of the cDNA obtained in step (10) as a template, the primer pair in step (1) as primers, and set up a no-template negative control for PCR reaction.

[0097] Experimental results: DNA gel electrophoresis results show (see Figure 5 ), in wild-type CHO-K1, all primer pairs could detect products; in cell line A, only primer pairs GS5-E1F and GS5-E2R could detect products, while primer pairs GS5-E2F and GS5-E3R, primer pairs GS5-E6F and GS5-E7R, and primer pairs GS1-F and GS1-R could not detect products; in cell line B, primer pairs GS5-E1F and GS5-E2R and primer pairs GS1-F and GS1-R could detect products.

[0098] Example 3: Verification of GS gene functional defect Wild-type CHO-K1 cell line, cell line A and cell line B were cultured in medium without glutamine, 1 mM glutamine, 2 mM glutamine, 4 mM glutamine and 8 mM glutamine, and cell viability and density were monitored daily. The initial inoculum cell density was 5x10 5 cells / mL, the volume is 10mL.

[0099] Experimental results (see Figure 6 ): In the culture medium without glutamine, the wild-type cell line, cell line A and cell line B could not survive normally, among which cell lines A and B died rapidly and massively within 2 days, and the wild-type cell line was almost completely dead after 4 days; in the culture medium with low concentration of glutamine (1mM and 2mM), the growth rate of cell line A and cell line B was slower than that of the wild-type cell line; in the culture medium with high concentration of glutamine, there was no difference in the growth rate and viability of the three groups of cell lines.

[0100] Example 4: Screening and application of GS cell-deficient subclone cell lines with high expression levels (1) Cell lines A and B were plated for monoclonal colonies by mouth pipette selection. 8 mM glutamine was added to the culture medium used for plating, and 100 μL of culture medium was added to each well.

[0101] (2) The cells were cultured in a 37°C, 5% CO2 incubator and the fluid was replenished every 3 days with 35 μL of fluid each time.

[0102] (3) The three subclones that first reach a cell confluence rate of more than 60% are selected and numbered A1, A2, A3, B1, B2 and B3. It usually takes 10 to 14 days for the confluence rate to reach 60%.

[0103] (4) The 6 subcloned cell lines in step (3) were expanded and subcultured from the 96-well plate to the 24-well plate. When the confluence rate reached about 80%, they were subcultured to T25 culture flasks. Finally, when the cell density reached 5x10 5 cells / mL, expand the culture to shake flasks and store them in cryopreserved form as required.

[0104] (5) The constructed recombinant protein expression vector carrying exogenous GS was transferred into A1, A2, A3, B1, B2 and B3 cell lines in the logarithmic growth phase. On the day of transfection, 300,000 cells were mixed with 1 μg of plasmid vector and electroporated using the Thermo Neon transfection system. The transfection conditions were 1400V / 10msec / 3pulses.

[0105] (6) The transfected cells were placed in a culture medium containing 4 mM glutamine and allowed to recover for 48 hours.

[0106] (7) After 48 hours, medium without glutamine was added for half-medium replacement screening culture. Cell viability and density were tested every other day, and the time required for cell viability recovery was recorded for each cell line.

[0107] (8) When the cell line viability reaches more than 90%, subculture it into a shake flask and culture it for more than three generations.

[0108] (9) The cell lines prepared in step (8) are plated for monoclonal proliferation. When the confluence rate of the monoclonal cell lines reaches more than 60%, ELISA is performed to select the three monoclonal cell lines with the best reactions, which are numbered A11, A12, A13, A21, A22, A23, A31, A32, A33, B11, B12, B13, B21, B22, B23, B31, B32 and B33.

[0109] (10) After the 18 cell lines in step (9) were expanded and cultured, 2×105 Cells / mL density was inoculated and cultured, and the cell density reached 1x10 6 When the cell count was 2.5447 cells / mL, feeding induction was started. Cytiva 7a (5%) and Cytiva 7b (0.5%) were added to maintain the glucose content in the culture medium within the range of 7 g / L. The cell density, viability and antibody expression were recorded every day.

[0110] (11) When the cell density stabilizes and the viability begins to drop to 80%, the feeding induction is stopped and the final expression level of the cell line is determined based on this.

[0111] Experimental results (see Figure 7 and Figure 8 ): Figure 7 The figure shows the required recovery time for screening of 18 monoclonal cell lines, among which the screening cycles of A12 (CHO-K1 / LG-GSKa1) and B21 (CHO-K1 / LG-GSKb2) are relatively short. Figure 8 The expression levels of recombinant proteins of the 18 monoclonal cell lines are shown in the figure. They all obtained a relatively high recombinant protein expression level. Among them, the recombinant protein expression levels of the 9 subclone cells of the A cell line were relatively higher than that of the B cell line. The overall advantages of the three subclones of A1 were obvious over the other cell lines, and A12 was the best cell line.

[0112] The above results show that the A1 monoclonal GS-deficient subclone cell line discovered was obtained by combining multiple methods such as screening cycles and recombinant protein expression tests on the GS-deficient cell line. It has the characteristics of high protein expression, no need for MSX screening, short screening cycle, and safety and reliability in production applications.

[0113] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0114] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A Chinese hamster ovary cell line with glutamine synthetase gene knockout, characterized in that: It was named Chinese Hamster Ovary Cell CHO-K1 / LG-GSKa1 and was deposited in the China Center for Type Culture Collection on May 29, 2024; the deposit number is CCTCCNO: C2024149.

2. The Chinese hamster ovary cell line according to claim 1, characterized in that: The Chinese hamster ovary cell CHO-K1 / LG-GSKa1 is a subclone cell strain in which the glutamine synthetase genes on chromosome 5 and chromosome 1 of the CHO-K1 cell strain are double knocked out.

3. The Chinese hamster ovary cell line according to claim 2, characterized in that: The knocked-out gene sequence on chromosome 5 is a 5385 bp sequence from intron 1 to exon 7 of the glutamine synthetase gene, as shown in SEQ ID NO.35; The knocked-out gene sequence on chromosome 1 is a 1325 bp long sequence of the glutamine synthetase gene including the entire exon, such as SEQ ID NO.

36.

4. Use of the Chinese hamster ovary cell line according to any one of claims 1 to 3 in recombinant protein expression.

5. A Chinese hamster ovary cell line with glutamine synthetase gene knockout, characterized in that: It was named Chinese Hamster Ovary Cell CHO-K1 / LG-GSKb2 and was deposited in the China Center for Type Culture Collection on May 29, 2024; the deposit number is CCTCCNO: C2024167.

6. The Chinese hamster ovary cell line according to claim 5, characterized in that: The Chinese hamster ovary cell CHO-K1 / LG-GSKb2 is a subclone cell line with double knockout of the glutamine synthetase gene on chromosome 5 of the CHO-K1 cell line.

7. The Chinese hamster ovary cell line according to claim 5, characterized in that: The knocked-out gene sequence on chromosome 5 is a 5385 bp sequence from intron 1 to exon 7 of the glutamine synthetase gene, as shown in SEQ ID NO.

35.

8. Use of the Chinese hamster ovary cell line according to any one of claims 5 to 7 in the expression of recombinant proteins.

9. A method for obtaining the Chinese hamster ovary cell line according to claim 1 or 5, characterized in that: include: 1) Analyze the glutamine synthetase gene on chromosomes 5 and 1 of CHO-K1 cells, develop a knockout plan, and design the Cas9-guided sgRNA sequence that recognizes the glutamine synthetase gene; 2) Connect the tracrRNA sequence to the sgRNA sequence and synthesize the PCR reaction primers; 3) PCR amplification to obtain the sgRNA in vitro transcription template; 4) Obtaining sgRNA containing sgRNA sequence and tracrRNA sequence by in vitro transcription with RNA polymerase; 5) Combine sgRNA with Cas9 protein to form RNP complex, and transfect into CHO-K1 cells; 6) Monoclonal culture to obtain CHO-K1 monoclonal cell line with glutamine synthetase gene knockout; 7) Expand the culture and cryopreservation of the CHO-K1 monoclonal cell line with glutamine synthetase gene knockout; 8) The RNA level, protein level and functional defect of glutamine synthetase in the CHO-K1 monoclonal cell line with glutamine synthetase gene knockout obtained in 7) were verified, and after screening, a CHO-K1 subclonal cell line with high expression of glutamine synthetase gene knockout was obtained.

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