Preparation method and application of glutamine synthetase diallele knockout CHO cell line

Abstract

The invention relates to a preparation method and application of a glutamine synthetase diallele knockout CHO cell line, and the preparation method comprises the following steps: using a specially designed homologous recombination targeting sequence, delivering the targeting sequence to a CHO cell nucleus through a recombinant adeno-associated virus, and knocking out a glutamine synthetase gene of a CHO cell through homologous recombination, so as to obtain the glutamine synthetase diallele knockout CHO cell line. A positive and negative screening system is used for screening non-specific targeting events to obtain the glutamine synthetase biallele knockout CHO cell line, and the nucleic acid sequence of the targeting sequence comprises a sequence as shown in SEQ ID NO.1. The invention also discloses a preparation method of the glutamine synthetase biallele knockout CHO cell line. The GS diallele knockout CHO cell line without resistance selection markers is constructed, the gene knockout targeting efficiency based on homologous recombination is remarkably improved, and the off-target effect and the positive clone screening efficiency are reduced.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a preparation method and application of a glutamine synthetase biallelic gene knockout CHO cell line. Background Art

[0002] Chinese Hamster Ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster. They are one of the main means of manufacturing therapeutic proteins in the biopharmaceutical industry and one of the main tools for producing monoclonal antibodies. They can quickly identify those rare high-yielding cell lines among a large number of low-yielding and non-productive cells, and achieve an effective and efficient clinical cell line development process, which is of great significance for accelerating the development of biological drugs.

[0003] In cell culture, glutamine is used for cell energy metabolism on the one hand, and participates in protein synthesis and nucleic acid metabolism on the other hand. Under culture conditions without or with low glutamine, glutamine synthetase (GS) catalyzes the synthesis of glutamate and ammonium ions to generate glutamine, which supplies cell metabolism and protein synthesis. Methionine sulfonate (MSX) is a competitive inhibitor of GS. After MSX binds to the glutamate site of GS, it will be phosphorylated by ATP, thereby irreversibly inhibiting GS activity. The GS / MSX screening system is widely used to screen protein drug engineering cell lines based on CHO cells. Usually, the target protein gene and GS gene are co-transfected into the cell, and the GS gene is used as a screening marker gene to express glutamine synthetase, so that positive cells can grow under low or no glutamine conditions. Subsequently, by gradually increasing the concentration of MSX, cells with high copies and high expression of GS genes in the cell population are screened. These cells generally also carry a higher copy number of the target gene and can highly express the target gene protein. In addition, the engineered cell lines constructed using the GS / MSX method do not require the addition of glutamine during culture, which greatly reduces the accumulation of metabolic waste - ammonia in the culture medium. It has the advantages of easy optimization of the culture process, easy quality control, and high protein expression.

[0004] Industrial production of recombinant antibodies usually selects CHO cell lines with the endogenous GS gene knocked out as host cells. Although these cells cannot grow in a culture medium lacking glutamine, the GS gene carried on the exogenous plasmid can not only increase the rigor of screening stable cell lines in a culture medium lacking glutamine, but also enhance the reaction of converting waste ammonium ions into glutamine amino acids, which is very beneficial for large-scale cell culture.

[0005] At present, there are four main biotechnologies that can be maturely applied to GS gene knockout: homologous recombination, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and prokaryotic acquired immune system (clustered regularly interspaced short palindromic repeats, CRISPR). The traditional homologous recombination method constructs a gene targeting vector and uses the recombination of the left and right homologous arms to achieve the replacement of the target gene fragment with an exogenous gene. Although this method is easy to implement, the targeting efficiency is very low. In theory, homologous recombination events only occur in a short period of time during the replication of the host cell genome. According to statistics, the efficiency of homologous recombination in mouse embryonic stem cells (ES) is only one in a million, and the efficiency of homologous recombination in CHO cells is even much lower than that of ES cells. The successful invention and improvement of ZFN, TALEN, and CRISPR technologies have greatly facilitated researchers to perform subtle and precise operations at the genome level on prokaryotic cells, eukaryotic cells, etc. In terms of construction time and knockout efficiency, the three major editing technologies of ZFN, TALEN and CRISPR are obviously more advantageous than traditional homologous recombination. However, these three methods are still limited by their principles and mechanisms, and all have different degrees of off-target effects that cannot be ignored. In short, these three methods only rely on very short guide units or guide sequences to locate near the target sequence and complete the genome cutting under the action of nucleases, which can easily cause nucleases to cut non-target areas due to mismatches. For the CHO cell genome alone, for a standard GN19NGG target sequence, there are an average of 6 identical sequences, 377 sequences with one nucleotide mismatch, and 4558 sequences with two nucleotide mismatches in the CHO-K1 genome. Transgenic mice obtained by site-directed gene modification using CRISPR technology even have up to 1,397 single nucleotide mutations and 117 insertions or deletions of DNA sequences at the whole genome level. With so many genetic and epigenetic uncertainties, the knockout cell lines produced by this method are difficult to use as universal engineering cell lines for the production of medical proteins, and it is difficult to obtain approval from the National Medical Products Administration and the U.S. Food and Drug Administration.

[0006] CN116716253A discloses a GS gene knockout CHO cell line and its construction method and application. The construction method of the GS gene knockout CHO cell line adopts sgRNA truncated to 17bp, and its knockout efficiency is slightly improved compared with the traditional 20bp sgRNA, but it fails to fundamentally solve or avoid how to reduce the off-target effect of CRISPR technology.

[0007] In summary, how to provide a method that has advantages in safety, knockout accuracy, off-target effect, knockout efficiency and screening efficiency, and is truly accepted by the biopharmaceutical industry and applied to the actual transformation of CHO cell engineering strains has become one of the urgent problems to be solved in this field. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a preparation method and application of a glutamine synthetase double allele knockout CHO cell line. The method provided by the present invention greatly improves the targeting efficiency of traditional homologous recombination and significantly reduces the probability of non-specific integration of traditional homologous recombination. At the same time, the knockout positive cell line screening method provided by the present invention can easily obtain a double allele knockout positive CHO cell line of glutamine synthetase without resistance screening marker residue, improve the screening efficiency, and the cell line obtained by knockout has fast multiplication, high density, high yield, and the stable cell pool can reach the g / L level.

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a homologous recombination targeting sequence, wherein the nucleic acid sequence of the homologous recombination targeting sequence includes the sequence shown in SEQ ID NO.1.

[0011]

[0012] The homologous recombination targeting sequence used in the present invention comprises a left homology arm, a LoxP sequence, an SV40 promoter, a NeoR sequence, a LoxP sequence, a right homology arm, an SV40 promoter and an HSV-TK sequence, wherein the length of the left homology arm is 3 times of the target gene deletion fragment, the length of the right homology arm is 4 times of the target gene deletion fragment, the target gene deletion fragment is located in the sixth exon region of the CHO cell glutamine synthetase gene, the base length of the target gene deletion fragment is not an integral multiple of 3, the LoxP sequence is a recognition site of the Cre recombinase, the NeoR sequence is a positive screening sequence, and the HSV-TK sequence is a negative screening sequence.

[0013] In a second aspect, the present invention provides a method for preparing a glutamine synthetase bi-allelic knockout CHO cell line, the preparation method comprising: using the homologous recombination targeting sequence described in the first aspect to knock out the glutamine synthetase gene of the CHO cell.

[0014] Preferably, the DNA of the targeting sequence is contained in a recombinant adeno-associated virus vector.

[0015] Preferably, the recombinant adeno-associated virus vector is a vector produced by combining the AAV2 genome and the capsid protein.

[0016] Preferably, the preparation method specifically comprises the following steps:

[0017] (1) using a recombinant adeno-associated virus to deliver the targeting sequence into the nucleus of a CHO cell, causing the targeting sequence to undergo specific homologous recombination with the target fragment in the sixth exon region of the glutamine synthetase gene, thereby knocking out the glutamine synthetase gene of the CHO cell, using a positive and negative screening system to screen for non-specific targeting events, using Cre recombinase to remove the screening marker, and using qPCR to detect the integration and removal frequency of the screening marker, thereby obtaining a glutamine synthetase monoallelic knockout CHO cell line;

[0018] (2) Repeat step (1) and use the glutamine cell proliferation comparison experiment to quickly screen the double knockout probability events, and finally obtain the glutamine synthetase double allele knockout CHO cell line.

[0019] The present invention adopts a three-party combined screening strategy of delivering the above-mentioned targeting sequence into the nucleus for homologous recombination mediated by recombinant adeno-associated virus vector rAAV, site-specific recombinase Cre / LoxP system and positive and negative screening system to construct a GS double allelic knockout CHO cell line without resistance screening marker, significantly improves the gene knockout targeting efficiency based on homologous recombination and significantly reduces non-specific integration events, and is easier to obtain a residual glutamine synthetase double allelic knockout positive CHO cell line without resistance screening marker, thereby more effectively avoiding the potential risks brought by non-specific integration of the targeting sequence.

[0020] Preferably, the nucleic acid sequence of the sixth exon region of the glutamine synthetase gene includes the sequence shown in SEQ ID NO.2.

[0021] SEQ ID NO.2: tgggaattccaaataggaccctgtgaaggaatccgcatgggagatcatctctgggtggcccgtttcatcttgcat cgagtatgtgaagactttggggtaatagcaacctttgaccccaagcccattcctgggaactggaatggtgcaggctgccataccaactttagcac caaggccatgcggggggagaatggtctgaa.

[0022] Preferably, before obtaining the glutamine synthetase monoallelic knockout CHO cell line in step (1), a monoclonal cloning step is also included.

[0023] Preferably, before obtaining the glutamine synthetase biallelic knockout CHO cell line in step (2), a monoclonal cloning step is also included.

[0024] Preferably, before removing the screening marker and cloning, the method further comprises using fluorescent quantitative PCR to detect and analyze the clone ratio of the positively integrated targeting sequence in the cell pool and the clone ratio of the positively knocked-out target gene after the screening marker is removed.

[0025] Preferably, the nucleic acid sequences of the PCR primers used in the fluorescent quantitative PCR detection process include the sequences shown in SEQ ID NO.3 to SEQ ID NO.6.

[0026] SEQ ID NO. 3: ccggctacctgcccattcg.

[0027] SEQ ID NO. 4: tggcgaacagttcggctggc.

[0028] SEQ ID NO. 5: tctatgagggctacgctctcc.

[0029] SEQ ID NO. 6: acgctcggtcaggatcttca.

[0030] Preferably, the amino acid sequence of the Cre recombinase includes the sequence shown in SEQ ID NO.13.

[0031] SEQ ID NO.13: MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRML IHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD.

[0032] Preferably, before obtaining the glutamine synthetase biallelic knockout CHO cell line, the method further comprises obtaining genomic DNA of a monoclonal CHO cell line, and screening the glutamine synthetase biallelic knockout CHO cell line through sequencing data of the genomic DNA.

[0033] Preferably, the sequencing data is nested PCR sequencing data.

[0034] Preferably, the nucleic acid sequences of the nested PCR primers used in the process of obtaining the sequencing data include the sequences shown in SEQ ID NO.7 to SEQ ID NO.12.

[0035] SEQ ID NO.7: gctgtcttgtgaatagagggt.

[0036] SEQ ID NO. 8: catggcatgtagtttgtgagaa.

[0037] SEQ ID NO. 9: cagcgtgcagataggatgaa.

[0038] SEQ ID NO. 10: catggccttggtgctaaagtt.

[0039] SEQ ID NO. 11: ccttgtcattttctggctttg.

[0040] SEQ ID NO. 12: gtccgtattactgtggtgtg.

[0041] In a third aspect, the present invention provides a glutamine synthetase biallelic knockout CHO cell line, wherein the glutamine synthetase biallelic knockout CHO cell line is prepared by the preparation method of the glutamine synthetase biallelic knockout CHO cell line described in the second aspect.

[0042] Preferably, the cell line contains a nucleic acid molecule encoding a protein of interest.

[0043] Preferably, the protein of interest comprises an antibody.

[0044] Preferably, the nucleic acid molecule is free from the genome of the modified CHO cell line or is integrated into the genome.

[0045] Preferably, the glutamine synthetase biallelic knockout CHO cell line transiently expresses or stably expresses the target protein.

[0046] Preferably, the glutamine synthetase biallelic knockout CHO cell line is used for batch culture or fed-batch culture.

[0047] In a fourth aspect, the present invention provides the use of any one or a combination of at least two of the homologous recombination targeting sequence described in the first aspect, the method for preparing the glutamine synthetase biallelic knockout CHO cell line described in the second aspect, and the glutamine synthetase biallelic knockout CHO cell line described in the third aspect in expressing a target protein or preparing an engineered cell expressing a target protein.

[0048] Preferably, the protein of interest comprises an antibody.

[0049] In a fifth aspect, the present invention provides a method for expressing a target protein, the method comprising the following steps:

[0050] (1) introducing a nucleic acid molecule encoding the target protein into the glutamine synthetase biallelic knockout CHO cell line described in the third aspect;

[0051] (2) Cultivating the glutamine synthetase biallelic gene knockout CHO cell line to express the target protein.

[0052] Preferably, the protein of interest comprises an antibody.

[0053] Preferably, the introduction method in step (1) comprises transfection using a transfection reagent or electrofection.

[0054] Preferably, the transfection reagent comprises a lipid transfection agent.

[0055] Preferably, the nucleic acid molecule in step (1) is contained in an expression vector.

[0056] Preferably, the expression vector comprises a plasmid.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The present invention creatively provides a homologous recombination targeting sequence, which uses a left homologous arm 3 times the length of the target gene deletion fragment and a right homologous arm 4 times the length of the target gene deletion fragment to perform homologous targeting on the target sequence. The target gene deletion fragment is located in the sixth exon region of the glutamine synthetase gene of the CHO cell, and the base length of the target gene deletion fragment is not an integer multiple of 3. The above-mentioned targeting sequence is delivered into the nucleus by rAAV to increase the probability of homologous recombination. Under the three-party combined screening strategy of the site-specific recombinase Cre / LoxP system and the positive and negative screening system, fluorescent quantitative PCR, glutamine proliferation comparison dependence experiment and monoclonal cell nested PCR sequencing are used to improve the screening efficiency of GS knockout CHO cell lines. The method provided by the present invention adopts a specially designed homologous targeting sequence and delivers it into the nucleus through a recombinant adeno-associated virus, successfully solving the problem of low targeting efficiency of traditional homologous recombination. In the process of screening cell lines using a positive and negative screening system, false positive clones caused by non-specific targeting are screened out, so the off-target rate is low. The screening marker can be easily removed by the Cre recombinase system. The double allele knockout positive CHO cell line with no resistance screening marker and residual glutamine synthetase can be quickly obtained by fluorescent quantitative PCR and glutamine proliferation comparison dependence experimental methods. The cell line obtained by knockout has fast doubling, high density, high yield, and the stable cell pool can reach the g / L level. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the exon distribution and targeting design of the MeCHOta CHO-K1 GS coding gene;

[0060] Figure 2 The first homologous recombination targeted cell pool genotype qPCR detection result diagram in Example 1, wherein Figure a is a detection result diagram of the frequency of the targeting sequence integration event, and Figure b is a detection result diagram of the frequency of the screening marker sequence removal event;

[0061] Figure 3The graphs are the results of molecular identification of the genotype of the MeCHOta CHO-K1 GS monoallelic knockout cell line in Example 1, wherein graph a is the result of nucleic acid electrophoresis detection, and graph b is the result of sequencing;

[0062] Figure 4 The results of the screening marker antibiotic sensitivity verification of the glutamine synthetase biallelic knockout MeCHOta CHO-K1 GS-KO cell line in Example 2 are shown in Figure a, wherein Figure a is a graph showing the relationship between viable cell density and time, and Figure b is a graph showing the relationship between viability and time;

[0063] Figure 5 This is a graph evaluating the glutamine dependence of cell proliferation of the MeCHOta CHO-K1 GS-KO cell line with biallelic knockout of glutamine synthetase in Example 3, wherein FIG. a is a graph showing the relationship between viable cell density and time, and FIG. b is a graph showing the relationship between viability and time;

[0064] Figure 6 This is a statistical graph of live cell density in fed-batch culture of the recombinant antibody expressing strain of the MeCHOta CHO-K1 GS-KO cell line with biallelic knockout of glutamine synthetase in Example 4;

[0065] Figure 7 This is a statistical graph of the viable cell rate of the recombinant antibody expression strain of the MeCHOta CHO-K1 GS-KO cell line with biallelic knockout of glutamine synthetase in Example 4;

[0066] Figure 8 This is a statistical graph of the antibody production of the recombinant antibody expression strain of the MeCHOta CHO-K1 GS-KO cell line with biallelic knockout of glutamine synthetase in Example 4 in a fed-batch culture. DETAILED DESCRIPTION

[0067] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0068] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0069] In order to more easily understand the present invention, some terms are first defined. As used in the present invention, unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. All patents and publications mentioned in the present invention are incorporated into the present invention in their entirety by reference.

[0070] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli cells or Bacillus subtilis cells, fungal cells such as yeast cells or Aspergillus cells, insect cells such as S2 Drosophila cells or SF9 cells, and animal cells such as fibroblasts, CHO cells, COS cells, NS0 cells, Hela cells, BHK cells, HEK293 cells or human cells, etc.

[0071] As used herein, the term "suspension acclimation" means that adherent cells can grow normally in suspension culture after several subcultures in an adherent state. Under the conditions of suspension culture, cells can be cultured in multiple layers in a liquid space.

[0072] As used herein, the term "serum-free culture" refers to a culture method in which cells are cultured using a medium that does not contain serum.

[0073] As used herein, the term "glutamine synthetase (GS)" refers to an enzyme that can catalyze the synthesis of glutamine from glutamate and ammonium. The international system classification number of GS is EC6.3.1.2. In the genome of CHO cells, the nucleotide sequence encoding GS is known, and its exemplary nucleotide sequence can be found, for example, in NCBI accession number NW_003613921.1; positions 1430036-1435423.

[0074] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids, phagemids, artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1, bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0075] As used herein, the term "transient expression" refers to the expression of a gene encoding an exogenous protein that is free from the host cell genome (i.e., not integrated into the host cell genome) as an exogenous protein by the host cell. Transient expression can achieve efficient expression of exogenous proteins in a short period of time. However, as cells divide, copies of the exogenous gene are gradually lost, and the expression level of the exogenous protein is gradually reduced.

[0076] As used herein, the term "stable expression" means that the host cell expresses the gene encoding the exogenous protein integrated into the host cell genome as the exogenous protein. In the process of stable expression, the gene encoding the exogenous protein is stably inserted into the genome (e.g., chromosome) of the host cell, so that the host cell can stably express the exogenous protein for a long time.

[0077] As used herein, the term "cell pool" refers to a mixed cell pool produced after a gene encoding an exogenous protein is stably inserted into the genome of a host cell. The mixed cell pool can be further sorted into a minipool cell pool of smaller units, or sorted into a monoclonal clone of a single cell. In the common cell strain screening process in the industry, cell pool screening is the initial screening stage, followed by a mini pool screening stage or a monoclonal screening stage.

[0078] As used herein, the term "gene knockout" refers to editing a gene in a cell genome (e.g., inserting, replacing, and / or deleting the gene) so that the gene loses its original function (e.g., cannot express a functional protein). Various known molecular biology techniques (e.g., gene editing techniques using ZFN, TALEN, CRISPR / cas9 or NgAgo) can be used to edit genes in the cell genome. Gene knockout is not limited to completely deleting or removing the entire gene, as long as the gene loses its original function. For example, an exogenous DNA fragment can be inserted into the gene so that the gene cannot express a functional protein, or one or more bases can be inserted or deleted in the gene so that the gene undergoes a frameshift mutation to achieve knockout of the gene.

[0079] In some embodiments, the sequencing data is nested PCR sequencing data. As used herein, the term "nested PCR" is a variant of PCR that uses two pairs of PCR primers to amplify complete fragments. The first pair of PCR primers amplify fragments similar to ordinary PCR. The second pair of primers is called nested primers, which are combined inside the first PCR product so that the second PCR amplification fragment is shorter than the first amplification. The advantage of nested PCR is that if the first amplification produces an erroneous fragment, the probability of primer pairing and amplification on the erroneous fragment for the second time is extremely low. Therefore, the amplification of nested PCR is very specific.

[0080] As used herein, the term "fed-batch culture" also known as "fed-batch culture" refers to a culture method in which cells are inoculated into a certain amount of culture medium for culture, and then as nutrients are continuously consumed, new culture medium is continuously added to the system to continue culturing the cells (i.e., extending the culture time of the cells) until the entire culture process is completed.

[0081] Sequence information

[0082] The information of the sequences involved in the present invention is provided in Table 1 below.

[0083] Table 1

[0084]

[0085]

[0086] Unless otherwise specified, the molecular biology experimental methods used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0087] Example 1

[0088] This example is about the construction of glutamine synthetase GS double allele knockout CHO cell line.

[0089] In this example, the adherent cell line ECACC CHO-K1 purchased from the European Standard Cell Collection Center was first subjected to suspension acclimation. In short, ECACC CHO-K1 cells were thoroughly acclimated to the serum-free, constitutive medium CD CHO Fusion in a gradient acclimation manner. After the acclimated cell passage doubling time tended to be stable, monoclonal sorting was performed, and the serum-free culture, suspension cell line finally obtained was named MeCHOta CHO-K1 cell line. Subsequently, the GS gene was knocked out based on the MeCHOta CHO-K1 cell line, and the specific steps were as follows:

[0090] 1. Design of targeting sequences and packaging of rAAV vectors

[0091] The experimental object of this example is CHO cells, and the NCBI gene library number of the GS gene sequence used is NW_003613921.1; position 1430036-1435423. The GS gene contains 7 exons, of which the target sequence targeted by this example is located in the sixth exon region. The exon distribution and targeting design of the GS coding gene are as follows: Figure 1As shown. The sequence used for delivering the target specifically includes the left homology arm, LoxP sequence, SV40 promoter, NeoR sequence, LoxP sequence, right homology arm, SV40 promoter, and HSV-TK sequence in order. The left homology arm is 300bp in length, the right homology arm is 396bp in length, and the targeting sequence is named GSKO-PNS-S1, and its nucleotide sequence is shown in SEQ ID NO.1. NeoR in the targeting sequence is used as a positive screening marker gene for the positive and negative screening system for positive screening of sequence targeting. HSV-TK is used as a negative screening marker gene for the positive and negative screening system to screen out cells with non-specific integration of the targeting sequence in the CHO cell genome. The targeting sequence of the control group only includes the left homology arm, LoxP sequence, SV40 promoter, NeoR sequence, LoxP sequence, and right homology arm in order. The left homology arm is 1124bp in length, the right homology arm is 1872bp in length, and the targeting sequence is named GSKO-PS-S2.

[0092] The nucleic acid chain synthesis of the targeting sequence, the construction of the rAAV plasmid vector, the packaging and production purification of the rAAV virus, and the construction of the Cre recombinase expression plasmid (pCDNA3.1-Cre, the Cre nucleic acid sequence is inserted into the multiple cloning site of the pCDNA3.1 vector, and the amino acid sequence encoded by the Cre recombinase is shown in SEQ ID NO.13) were all outsourced to Suzhou Jinweizhi Biotechnology Co., Ltd.

[0093] 2. rAAV transfection mediated the first homologous recombination targeting

[0094] First, revive a well-trained MeCHOta CHO-K1 cell according to the standard cell culture process. According to the dilution subculture method, use the subculture medium (CD CHO Fusion + 6mM Gln) with a subculture density of 0.3×10 6 / mL, subculture volume 20mL, subculture once every three days. After three generations, adjust the density to 0.5×10 6 / mL, ready for rAAV transfection. The next day, the cell density grew to about 1×10 6 After that, 1×10 5 The host cells were plated into 24-well plates. Then, the cells were plated at an MOI of 10. -2 , MOI = 10 -1 , MOI = 1, take the corresponding amount of diluted virus solution and add it to the 24-well plate with cells, mix gently and evenly, and place it in the incubator for three days. The culture conditions are 37°C, 5% CO2, and 80% relative humidity.

[0095] 72h after transfection, collect all the cells in each experimental well into a 1.5mL centrifuge tube, discard the supernatant after centrifugation, and the centrifugation condition is 200g, 5min. Use an equal volume of screening medium (CD CHO Fusion + 8mM Gln + 0.33% ACA + 400μg / mL G418 + 25μg / mL GANC), resuspend the cell pellet, and continue to place it in the incubator for static culture after cell counting. Centrifuge according to the same steps every three days until the cell viability of each experimental group recovers to 90%, and then dilute and subculture according to the standard cell culture process.

[0096] 3. Removal of selection markers

[0097] The transfected cell pool in the previous step was treated by dilution subculture. Specifically, subculture medium (CD CHO Fusion + 8mM Gln + 400μg / mL G418) was used, and the subculture density was 0.3×10 6 / mL, subculture volume 20mL, subculture once every three days. After three generations, adjust the density to 0.5×10 6 / mL, in preparation for the electroporation of the Cre recombinase expression plasmid (pCDNA3.1-Cre). The next day, the cell density grew to about 1×10 6 After the cells were electroporated at 0.5×10 / mL, the CHO cells were electroporated according to the instructions of the Lonza Nucleofector 2b electroporator and the Lonza electroporation kit Cell line Nucleofector TMKit V. 6 The cells were inoculated at a density of 100 cells / mL, and then subcultured every three days using subculture medium (CD CHO Fusion+8mM Gln) until the cell viability recovered to more than 90%.

[0098] 4. qPCR detection of targeting sequence integration events and screening marker removal events

[0099] Take 1×10 cells from the cell pool for passage in the TPP tube. 7 The whole genome DNA was extracted according to the instruction of Takara MiniBESTUniversal Genomic DNA Extraction Kit Ver.5.0, and the product was used as the template DNA for subsequent qPCR identification.

[0100] qPCR primers were designed on the homologous recombination target sequence. The sequences of the qPCR primers included a detection primer pair with sequences shown in SEQ ID NO.3 and SEQ ID NO.4 and an internal reference primer pair with sequences shown in SEQ ID NO.5 and SEQ ID NO.6. Premix Ex Taq TM II kit instructions, quantitative detection of the target gene frequency, the test results are as follows Figure 2 As shown. Among them, Figure a shows that the target genotype frequency of the cell pool produced after targeting of the targeting sequence GSKO-PNS-S1 provided by the present invention accounts for about 70% of the total genotype, while the frequency of the control sequence is about 130%. Theoretically, when no specific integration event occurs, the frequency of the genotype detected after targeting should be 50%, which means that the targeting sequence of the present invention significantly improves non-specific integration events, and its effect is very close to 50%, while the frequency of the control is nearly twice that of the present invention, indicating that there are extremely large numbers of non-specific integration events in the cell pool. Figure 2 Figure b shows that the target genotype frequency of the cell pool produced by the targeting sequence GSKO-PNS-S1 provided by the present invention after the Cre recombinase is less than 20% of the total genotype, while the control sequence frequency exceeds 60%, indicating that the targeting sequence of the present invention is easier to completely remove the screening marker and reduce the residue than the control.

[0101] 5. Monoclonal Isolation

[0102] The cell pool from the previous step was subcultured according to the dilution subculture method, and the density was adjusted to 0.5×10 6 / mL, in preparation for single clone sorting. The next day, the cell density grew to about 1×10 6 After reaching 1 cell / mL, wash the cells to be sorted according to the instructions for use of the C.sight single-cell printer. Subsequently, add the treated cell suspension to the sample loading slot of the sorting chip. Plate 96-well plates with 1 cell per well. After all wells are plated, place them in the incubator for 2 hours, and use VIPs to take images of the sorted monoclones. Take images again on the 14th day of cell culture, and select monoclonal cells with a confluence greater than 70% for step-by-step transfer.

[0103] During the monoclonal transfer process, the screening efficiency of double knockout monoclones was further improved by comparing the glutamine proliferation. Specifically, during the recovery period of the 24-well plate, all monoclones were divided into two and cultured in a medium containing 8mM glutamine and a medium without glutamine, respectively. After culturing to the fifth day, the cells were sorted according to the ratio of the live cell density of the two culture conditions, and the top 15 monoclonal cell pools were selected from the largest to the smallest difference value, and then they were passaged normally step by step until they restored the stable doubling time and density.

[0104] 6. Genotype identification

[0105] Take 1×10 monoclonal cells recovered from the previous step and subcultured in the TPP tube 7 The whole genome DNA was extracted according to the instruction of Takara MiniBESTUniversal Genomic DNA Extraction Kit Ver.5.0, and the product was used as the template DNA for subsequent PCR identification.

[0106] Nested PCR primers are designed around the editing site, and the sequences of the nested PCR primers include a primer pair with sequences shown as SEQ ID NO.3 and SEQ ID NO.4 and a primer pair with sequences shown as SEQ ID NO.5 and SEQ ID NO.6.

[0107] Preparation of PCR reaction solution: In a 0.25mL PCR tube, add 2×Rapid Taq Master Mix 25μL, whole genome DNA template 1μL, 10μmol / L nested PCR primers, 1.5μL of each primer, and add water to 50μL PCR reaction system. PCR reaction conditions: pre-denaturation at 94℃ for 3min, denaturation at 95℃ for 30s, annealing at 55℃ for 30s, extension at 72℃ for 30s, cycle 35 times, and store at 4℃. Preparation of electrophoresis solution: Take all the solution after PCR reaction and use it for 1% agarose electrophoresis.

[0108] Nested PCR was used to identify monoclonal cells. The electrophoresis results of the PCR amplified fragments were as follows: Figure 3 As shown in Figure a, lanes 1-15 are clones 1B3, 1C2, 1C5, 1D7, 1F7, 1G7, 2B11, 2C11, 2E5, 2G6, 3E8, 3F3, 3H10, 4A2 and 4D7, and lane M is DL5000 Marker. The sequencing results are shown in Figure 3 As shown in Figure b, the sequencing peaks of clones 1B3, 1C2, 1C5, 1D7, 1F7, 1G7, 2C11, 2E5, 2G6, 3E8, 3F3, 3H10, 4A2 and 4D7 show nested peaks, and there are obvious insertion mutations at the sixth exon, so clones 1B3, 1C2, 1C5, 1D7, 1F7, 1G7, 2C11, 2E5, 2G6, 3E8, 3F3, 3H10, 4A2 and 4D7 are identified as GS monoallelic knockout positive clones and named MeCHOta CHO-K1 GS- / + cell line. The above results show that the knockout positive rate of the technical effect of the present invention exceeds 93%, far exceeding the screening probability of 1 / 1000000 of the initial homologous recombination method.

[0109] 7. Construction of Glutamine Synthetase GS Bi-allelic Knockout CHO Cell Line

[0110] According to the cell state, MeCHOta CHO-K1 GS- / + cell line 2C11 clone was selected as the host cell for further knockout. The knockout steps were rAAV transfection-mediated second homologous recombination targeting, removal of the screening marker, monoclonal sorting and genotype identification. The specific operation steps were carried out with reference to steps 2-6 of this embodiment. The GS double allele knockout positive clone after screening was named MeCHOta CHO-K1 GS-KO cell line.

[0111] Example 2

[0112] Validation of antibiotic susceptibility of selection markers for MeCHOta CHO-K1 GS-KO.

[0113] MeCHOta CHO-K1 GS-KO cells were cultured at 5 × 10 5 The cells were inoculated at a density of 100 cells / mL into CD CHO Fusion medium supplemented with 8 mM glutamine and 400 μg / mL G418, and subcultured at 37°C in suspension conditions. The cells were subcultured in equal volumes every three days, and the cells were counted before and after subculture. Figure 4 As shown in the figure, Figure a shows the relationship between live cell density and time, and Figure b shows the relationship between live cell density and time. On the third day, the live cell density of MeCHOta CHO-K1 GS-KO showed slow proliferation, and its live cell rate had dropped to 75%. After one passage, on the sixth day, its live cell density and live cell rate had dropped to 0. The experimental results show that the NeoR selection markers inserted in the construction process of the MeCHOta CHO-K1 GS-KO cell line have been removed, and it no longer has Neo resistance.

[0114] Example 3

[0115] Assessment of glutamine dependence of cell proliferation in the MeCHOta CHO-K1 GS-KO cell line.

[0116] MeCHOta CHO-K1 GS-KO were cultured in CD CHO Fusion medium containing high concentration of glutamine (8 mM) or without glutamine (0 mM). 6 Cells were inoculated at 100 / mL and cultured for 6 days, with cell density measured every day. Figure 5As shown, Figure a is a graph showing the relationship between live cell density and time, and Figure b is a graph showing the relationship between viability and time. The cell density and viability of MeCHOtaCHO-K1 GS-KO under different culture medium conditions are quite different. The results show that MeCHOta CHO-K1GS-KO can rapidly double in a culture medium containing high concentrations of glutamine (8mM), and its viability can be maintained at more than 96% from beginning to end. Under the conditions of a culture medium without glutamine (0mM), the growth of MeCHOta CHO-K1GS-KO cells almost completely stagnated, no longer doubled, and the viability showed a downward trend with the number of culture days. This indicates that the GS biallelic genes of MeCHOta CHO-K1 GS-KO cells cannot be expressed normally and function. After supplementing with sufficient exogenous glutamine, MeCHOta CHO-K1 GS-KO cells can resume normal proliferation. This shows that MeCHOta CHO-K1 GS-KO is a cell proliferation glutamine-dependent cell, and its endogenous GS gene has been successfully knocked out.

[0117] Example 4

[0118] Stable expression of foreign proteins in MeCHOta CHO-K1 GS-KO.

[0119] The expression plasmids encoding the test antibody molecules T-mAb and T-BsAb were electroporated into MeCHOtaCHO-K1 GS-KO cells in the logarithmic growth phase. After 48 hours of electroporation, the cells were transferred to a medium supplemented with 20 μM MSX and without glutamine, and the cells were cultured at 0.5×10 6 The cells were plated in 6-well plates at a density of 100 cells / mL. Then, the cells were cultured in 6-well plates with equal volume of medium change every three days until the cell density and viability reached the standard and then transferred to TPP tubes for dilution and subculture.

[0120] The cells that eventually recovered their normal doubling time were subjected to fed-batch evaluation. Specifically, all cells to be compared were adjusted to a density of 0.7×10 6 / mL, and the expression medium was Actipro medium. In addition, feed was added on the 3rd, 5th, 7th, 9th, 11th and 13th days of culture. During the culture period, the cell density was recorded. When the cell density reached 1×10 7 When the number of cells / mL was 40, the culture temperature was lowered from 37°C to 34°C for expression culture. After the culture was completed, the antibody level in the culture supernatant was measured using the biomolecular interaction instrument fortebio and protein A probe. During the fed-batch culture, the live cell density of the cell line was as follows Figure 6 As shown, the highest viable cell density of each cell line was 14.9×10 6 -23.9×106 The cell line viability results are as follows Figure 7 As shown in Figure 2, the cell viability of each cell line was maintained above 90% in the first 11 days of the fed-batch culture process. Figure 8 As shown, the maximum yield of monoclonal antibodies reached 1g / L, and the maximum yield of bispecific antibodies reached 500mg / L. These results show that the obtained MeCHOta CHO-K1 GS-KO performed excellently in the fed-batch culture process. Even in the initial cell pool stage of cell line screening, it can maintain the advantages of high cell density, high cell viability and high yield during the fed-batch culture process. Therefore, MeCHOta CHO-K1 GS-KO has a high application potential as a host cell line for expressing antibodies, meeting the needs of industrial large-scale production of recombinant antibody drugs.

[0121] In addition, it should be pointed out that the above results are only an evaluation of the cell pool screening stage. Researchers in this field can clearly see that the antibody production of these knockout cell lines can be further significantly improved by sorting monoclonal cells from the cell pool, optimizing the process flow, and expressing different types and types of antibody molecules.

[0122] The above results show that when MeCHOta CHO-K1 GS-KO is used to construct a cell population / cell line that stably expresses exogenous proteins (such as antibodies), it is easier to obtain a positive cell pool (especially a positive cell population with high exogenous protein production), and the exogenous protein (such as antibody) production of the obtained positive cell pool also significantly reaches the g / L level.

[0123] In summary, the present invention creatively provides a homologous recombination targeting sequence, and adopts a three-party combined screening strategy of rAAV delivery of the targeting sequence into the nucleus to mediate homologous recombination, site-specific recombinase Cre / LoxP system, and positive and negative screening system. Through fluorescence quantitative PCR and glutamine increment comparison experiments, the gene knockout targeting efficiency based on homologous recombination is significantly improved, the off-target effect and screening efficiency are reduced, and a GS double allele knockout CHO cell line without resistance screening markers is successfully constructed. And the MeCHOta CHO-K1 GS-KO cell line obtained by this method has great potential in the application of expressing antibodies, providing a strong candidate engineering cell line for the field of biomedical CHO cells producing therapeutic recombinant antibodies.

[0124] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A homologous recombination targeting sequence, characterized in that: The nucleic acid sequence of the homologous recombination targeting sequence includes the sequence shown in SEQ ID NO.

1.

2. A method for preparing a glutamine synthetase biallelic gene knockout CHO cell line, characterized in that: The preparation method comprises: using the homologous recombination targeting sequence according to claim 1 to knock out the glutamine synthetase gene of CHO cells.

3. The method for preparing a glutamine synthetase biallelic knockout CHO cell line according to claim 2, characterized in that: The DNA of the targeting sequence is contained in a recombinant adeno-associated virus vector; Preferably, the recombinant adeno-associated virus vector is a vector produced by combining the AAV2 genome and the capsid protein.

4. The method for preparing a glutamine synthetase biallelic knockout CHO cell line according to claim 2 or 3, characterized in that: The preparation method specifically comprises the following steps: (1) using a recombinant adeno-associated virus to deliver the targeting sequence into the nucleus of a CHO cell, causing the targeting sequence to undergo specific homologous recombination with the target fragment in the sixth exon region of the glutamine synthetase gene, thereby knocking out the glutamine synthetase gene of the CHO cell, using a positive and negative screening system to screen for non-specific targeting events, using Cre recombinase to remove the screening marker, and using qPCR to detect the integration and removal frequency of the screening marker, thereby obtaining a glutamine synthetase monoallelic knockout CHO cell line; (2) Repeat step (1) and use the glutamine cell proliferation comparison experiment to quickly screen the double knockout probability events, and finally obtain the glutamine synthetase double allele knockout CHO cell line; Preferably, the nucleic acid sequence of the sixth exon region of the glutamine synthetase gene includes the sequence shown in SEQ ID NO.2; Preferably, before obtaining the glutamine synthetase monoallelic knockout CHO cell line in step (1), a monoclonal cloning step is also included; Preferably, before obtaining the glutamine synthetase biallelic gene knockout CHO cell line in step (2), a monoclonal cloning step is also included; Preferably, the amino acid sequence of the Cre recombinase includes the sequence shown in SEQ ID NO.

13.

5. The method for preparing a glutamine synthetase biallelic knockout CHO cell line according to claim 4, characterized in that: Before obtaining the glutamine synthetase biallelic gene knockout CHO cell line, the method further includes obtaining genomic DNA of a monoclonal CHO cell line, and screening the glutamine synthetase biallelic gene knockout CHO cell line through sequencing data of the genomic DNA; Preferably, the sequencing data is nested PCR sequencing data; Preferably, the nucleic acid sequences of the nested PCR primers used in the process of obtaining the sequencing data include the sequences shown in SEQ ID NO.7 to SEQ ID NO.

12.

6. A glutamine synthetase biallelic knockout CHO cell line, characterized in that: The glutamine synthetase biallelic gene knockout CHO cell line is prepared by the method for preparing the glutamine synthetase biallelic gene knockout CHO cell line according to any one of claims 2 to 5.

7. The glutamine synthetase biallelic knockout CHO cell line according to claim 6, characterized in that: The cell line contains a nucleic acid molecule encoding a target protein; Preferably, the target protein comprises an antibody; Preferably, the nucleic acid molecule is free from the genome of the modified CHO cell line or is integrated into the genome; Preferably, the glutamine synthetase biallelic knockout CHO cell line transiently expresses or stably expresses the target protein; Preferably, the glutamine synthetase biallelic knockout CHO cell line is used for batch culture or fed-batch culture.

8. Use of any one or a combination of at least two of the homologous recombination targeting sequence according to claim 1, the method for preparing a glutamine synthetase biallelic knockout CHO cell line according to any one of claims 2 to 5, and the glutamine synthetase biallelic knockout CHO cell line according to claim 6 or 7 in expressing a target protein or preparing an engineered cell expressing a target protein; Preferably, the protein of interest comprises an antibody.

9. A method for expressing a target protein, characterized in that: The method for expressing the target protein comprises the following steps: (1) introducing a nucleic acid molecule encoding the target protein into the glutamine synthetase biallelic knockout CHO cell line according to claim 6 or 7; (2) Cultivating the glutamine synthetase biallelic gene knockout CHO cell line to express the target protein.

10. The method for expressing a target protein according to claim 9, characterized in that: The target protein includes an antibody; Preferably, the method of introduction in step (1) comprises transfection using a transfection reagent or electrofection; Preferably, the transfection reagent comprises a lipid transfection agent; Preferably, the nucleic acid molecule in step (1) is contained in an expression vector; Preferably, the expression vector comprises a plasmid.

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