A DNA vector, a method for site-specific insertion of an exogenous gene sequence and application thereof

Abstract

The application discloses a DNA carrier, a method for site-specific insertion of an exogenous gene sequence and application thereof. The DNA carrier comprises a basic framework DNA carrier and a target gene DNA carrier. Genetic information of the basic framework DNA carrier comprises a first promoter gene sequence, a replaceable gene sequence, a second screening marker gene sequence and a second terminator gene sequence. The target gene DNA carrier comprises a target gene DNA expression unit. The target gene DNA expression unit comprises FRT3, a target gene sequence, a first terminator gene sequence, a second promoter gene sequence and FRT. The replaceable gene sequence can be replaced by the target gene DNA expression unit. The DNA carrier provided by the application can be used for realizing stable and efficient expression of an exogenous gene, and has a clear genetic background. Construction time of a stable cell strain is greatly shortened, and large-scale production and application of cell expression of an antibody or protein are promoted.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a DNA vector, a method for site-specific insertion of exogenous gene sequences, and their applications. Background Technology

[0002] Stable cell lines are cell lines in which a specific gene has been integrated into the cell genome using molecular biology methods, enabling the cells to stably express the target gene or interfere with gene expression for a long period. They are suitable for various research applications, such as recombinant protein and antibody production, gene editing, functional studies, and drug screening. Establishing stable cell lines can significantly reduce the cost of frequent transfections or viral packaging, and facilitate the study of gene function in target cells. Unlike transient transfection, which randomly introduces high copy numbers of expression in a short period, stable cell lines can be screened and amplified to obtain cell lines with stable high expression or appropriate copy numbers.

[0003] Methods for constructing stable transgenic cell lines can be broadly categorized into four types. The most mainstream method is lentivirus-mediated transfection, which boasts high mediation efficiency and short construction time. However, it carries certain biosafety risks, and packaging larger genes with lentiviruses is challenging. Cell lines may undergo apoptosis or mutation during lentivirus infection, and high-throughput stable transfection of multiple genes is labor-intensive. The second type is transposon system-mediated cell line stable transfection systems. These methods are safe and simple, facilitating transposition to transcriptionally active regions of the host genome. They can effectively mediate larger genes and sustain the expression of single or multiple genes, effectively compensating for the limitations of lentivirus-mediated transfection. The third type is the CRISPR knock-in system. This method enables site-specific insertion of exogenous genes. However, gene knock-in in CRISPR systems is a probabilistic event, and the knock-in efficiency varies depending on the sgRNA site selection and the inserted gene. Optimizing the sequence and mediation efficiency is necessary to improve the knock-in level, and significant time is required for single-clone sorting to obtain the correct site-specific knock-in cell lines. The fourth type is the most primitive method for constructing stable cell lines, which involves plasmid screening to select stable cell lines. The target plasmid is transfected into cells, and through long-term resistance screening, there is a certain probability of obtaining stable cell lines. However, due to its extremely low efficiency, it is rarely used nowadays. There are many methods for constructing stable cell lines, most of which involve random insertion; targeted insertion is much more difficult. Summary of the Invention

[0004] The main objective of this invention is to provide a DNA vector, a method for site-specific insertion of exogenous gene sequences, and its application, aiming to solve the problem of difficulty in site-specific insertion of target genes when preparing stable cell lines.

[0005] To achieve the above objectives, this invention proposes a DNA vector for site-specific insertion of exogenous gene sequences, wherein the DNA vector comprises:

[0006] A basic DNA vector includes a basic DNA expression unit, wherein the basic DNA expression unit comprises, from 5' to 3', a 5' homology sequence, a first promoter gene sequence, a replaceable gene sequence, a second selectable marker gene sequence, a second terminator gene sequence, and a 3' homology sequence.

[0007] The target gene DNA vector includes a target gene DNA expression unit, wherein the target gene DNA expression unit includes FRT3, the target gene sequence, the first terminator gene sequence, the second promoter gene sequence, and FRT in sequence from 5' to 3'.

[0008] The replaceable gene sequence includes FRT3, a first screening marker gene sequence, a first terminator gene sequence, and FRT in sequence from 5' to 3', and the replaceable gene sequence can be replaced by the target gene DNA expression unit by the FLP / FRT method.

[0009] Optionally, the first and second selection marker gene sequences are each selected from one of the hygromycin gene sequence or the glutamine synthase gene sequence; and / or,

[0010] The first promoter gene sequence and the second promoter gene sequence are each selected from one of the CMV promoter gene sequence or the SV40 promoter gene sequence; and / or,

[0011] The first terminator gene sequence and the second terminator gene sequence are respectively selected from either the β-globin polyA gene sequence or the SV40 polyA gene sequence.

[0012] Optionally, the first promoter gene sequence is a CMV promoter gene sequence, and the basic DNA expression unit further includes a CMV enhancer gene sequence, wherein the CMV enhancer gene sequence is located between the 5' end homology sequence and the first promoter gene sequence; and / or,

[0013] The basic DNA expression unit further includes an intron A gene sequence and an intron B gene sequence, wherein the intron A gene sequence is located between the first promoter gene sequence and FRT3, and the intron B gene sequence is located between FRT3 and the first selection marker gene sequence.

[0014] Optionally, the basic DNA vector is obtained by modifying the pUC19 plasmid. The basic DNA expression unit is constructed on the pUC19 plasmid for replication and amplification. A large number of linearized fragments are obtained by plasmid enzyme digestion to obtain the basic DNA vector; and / or,

[0015] The target gene DNA vector is obtained by modifying the pUC19 plasmid. The target gene DNA vector is constructed by inserting the target gene DNA expression unit into the pUC19 plasmid.

[0016] Optionally, the nucleotide sequence of the basic architecture DNA expression unit is as shown in SEQ ID NO: 1; and / or,

[0017] The nucleotide sequence of the target gene DNA expression unit is shown in SEQ ID NO: 2.

[0018] This invention also proposes a method for site-specific insertion of exogenous gene sequences, using the DNA vector described above, comprising the following steps:

[0019] S10. Select the cell line and genomic site to be modified by targeted insertion, and design sgRNA according to the site and the gene editing protein used. Construct the sgRNA on the plasmid to obtain the sgRNA plasmid.

[0020] S20. Using sgRNA plasmid, the basic DNA expression unit was electroporated into the cell line to be modified. Intermediate recombinant cell line was obtained through first antibiotic selection and single-clone selection.

[0021] S30. Using the FLP / FRT recombinase system, the target gene DNA expression unit with the target gene is inserted into the intermediate recombinant cell line. Through the second resistance screening, a cell line with a site-directed single copy of the target gene is obtained.

[0022] The cell line to be modified is a cell line that does not contain the second resistance selection gene.

[0023] Optionally, the step of "obtaining intermediate recombinant cell lines through first antibiotic screening and monoclonal screening" includes:

[0024] The basic DNA expression unit was determined to be inserted into the genome through primary antibiotic selection; the basic DNA expression unit was correctly inserted into the locus by genome sequencing; the basic DNA expression unit was inserted as a single copy by measuring the red-green light ratio through simultaneous electroporation of red and green fluorescent genes; and intermediate recombinant cell lines were obtained through monoclonal selection.

[0025] Optionally, the second screening marker is glutamine synthase, and the cell line to be modified is Chinese hamster ovary cells with glutamine synthase bis-allelic gene knockout.

[0026] Optionally, the sgRNA is one of sgRNA1, sgRNA2, and sgRNA3:

[0027] The sgRNA1 includes sgRNA1-F and sgRNA1-R, the gene sequence of sgRNA1-F is shown in SEQ ID NO: 3, and the gene sequence of sgRNA1-R is shown in SEQ ID NO: 4;

[0028] The sgRNA2 includes sgRNA2-F and sgRNA2-R, the gene sequence of sgRNA2-F is shown in SEQ ID NO: 5, and the gene sequence of sgRNA2-R is shown in SEQ ID NO: 6;

[0029] The sgRNA3 includes sgRNA3-F and sgRNA3-R, the gene sequence of which is shown in SEQ ID NO: 7, and the gene sequence of which is shown in SEQ ID NO: 8.

[0030] The present invention also proposes the application of the DNA vector described above in recombinant protein expression or gene function research.

[0031] In the technical solution of this invention, the DNA vector includes a basic DNA vector and a target gene DNA vector. The basic DNA vector includes a basic DNA expression unit, and the target gene DNA vector includes a target gene DNA expression unit. The basic DNA expression unit includes a replaceable gene sequence, which can be replaced by a DNA expression unit containing a target gene or a foreign gene through gene editing, thereby achieving the insertion of the target gene or the foreign gene. Since the basic DNA expression unit does not contain a promoter for the second selection marker gene, but when the target gene DNA expression unit is inserted into the basic DNA expression unit to replace the replaceable gene sequence, the resulting vector contains the promoter for the second selection marker gene, thereby enabling the expression of the second selection marker. This allows for the screening of vectors that have not successfully inserted into a specific site of the basic DNA expression unit, resulting in vectors that have successfully inserted the target gene or a foreign gene into a specific site of the basic DNA expression unit. Therefore, by inserting the aforementioned basic DNA expression unit into the reserved insertion site in the cell, and then inserting the target gene DNA expression unit into the specific site of the basic DNA expression unit through gene editing, it is possible to obtain stable cell lines with targeted insertion with a higher probability, and reduce the screening operations after the insertion of the target gene, greatly shortening the screening time after the insertion of the target gene, and quickly realizing the production and research applications related to the target gene. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0033] Figure 1 A schematic diagram of genetic information in one embodiment of the DNA vector, the basic architecture provided by the present invention;

[0034] Figure 2 A schematic diagram of genetic information in one embodiment of the target gene DNA vector provided by the present invention;

[0035] Figure 3 for Figure 2 Insertion of target gene DNA vector Figure 1 A schematic diagram of the genetic information following the basic DNA vector structure;

[0036] Figure 4 This is a schematic diagram of the chromosome walking gel running results in Example 1 of the present invention;

[0037] Figure 5 This is a schematic diagram of the sgRNA verification results in Example 2 of the present invention.

[0038] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0041] Stable cell lines are cell lines in which specific genes have been integrated into the cell genome using molecular biology methods, enabling the cells to stably express the target gene or interfere with gene expression for a long period. They are suitable for various research applications, such as recombinant protein and antibody production, gene editing, functional studies, and drug screening. Establishing stable cell lines significantly reduces the cost of frequent transfections or viral packaging, facilitating the study of gene function in target cells. Unlike transient transfection, which randomly introduces high copy numbers of expression in a short period, stable cell lines can be obtained through screening and amplification to achieve stable high expression or appropriate copy numbers. Methods for constructing stable cell lines can be broadly categorized into four types, with lentivirus-mediated methods being the most mainstream. These methods offer advantages such as high mediation efficiency and short construction time, but they also carry certain biosafety risks. Furthermore, packaging large genes with lentiviruses is challenging, and cell lines may undergo apoptosis or mutation during lentivirus infection. Additionally, high-throughput stable transfection of multiple genes is a labor-intensive process. The second type is the transposon system-mediated stable cell translocation system. This method is safe and simple, facilitates transposition to transcriptionally active regions of the host genome, and can effectively mediate the expression of larger genes, allowing for the sustained expression of single or multiple genes. This method effectively compensates for the limitations of lentivirus-mediated translocation. The third type is the CRISPR knock-in system. This method enables site-specific insertion of exogenous genes. However, gene knock-in in the CRISPR system is a probabilistic event, and the knock-in efficiency varies depending on the sgRNA site selection and the inserted gene. Optimizing the sequence and mediation efficiency is necessary to improve the knock-in level, and significant time is required for single-clone sorting to obtain the correct knock-in cell lines. The fourth type is the most primitive method for constructing stable transgenic cells: plasmid screening. The target plasmid is transfected into cells, and through long-term resistance screening, there is a certain probability of obtaining stable transgenic cell lines. Due to its extremely low efficiency, this method is rarely used nowadays. There are many methods for constructing stable transgenic cells, most of which involve random insertion, while site-specific insertion is quite difficult.

[0042] Currently, this is mainly achieved through the CRISPR knock-in system. However, directly designing sgRNA at selected sites on the genome carries a certain probability of insertion at other sites, and the number of inserted copies is not fixed, which may pose potential risks. Furthermore, a long period of single-clone screening and validation is still required to obtain stable transgenic strains with specific sites and copy numbers, which hinders its rapid application.

[0043] In view of this, the present invention proposes a DNA vector to solve the problem of difficulty in site-specific insertion of target genes when preparing stable cell lines.

[0044] In the technical solution of this invention, please refer to Figures 1 to 3The DNA vector includes a basic DNA vector and a target gene DNA vector. The basic DNA vector includes a basic DNA expression unit, which, from 5' to 3', sequentially includes a 5' homology sequence, a first promoter gene sequence, a replaceable gene sequence, a second selection marker gene sequence, a second terminator gene sequence, and a 3' homology sequence. The target gene DNA vector includes a target gene DNA expression unit, which, from 5' to 3', sequentially includes FRT3, the target gene sequence, the first terminator gene sequence, the second promoter gene sequence, and FRT. The replaceable gene sequence, from 5' to 3', sequentially includes FRT3, the first selection marker gene sequence, the first terminator gene sequence, and FRT, and the replaceable gene sequence can be replaced by the target gene DNA expression unit.

[0045] In the technical solution of this invention, the DNA vector includes a basic DNA vector and a target gene DNA vector. The basic DNA vector includes a basic DNA expression unit, and the target gene DNA vector includes a target gene DNA expression unit. The basic DNA expression unit includes a replaceable gene sequence, which can be replaced by a target gene DNA expression unit containing a target gene or a foreign gene through gene editing methods, thereby achieving the insertion of the target gene or the foreign gene. Since the basic DNA expression unit does not contain a promoter for the second selection marker gene, but when the target gene DNA expression unit is inserted into the basic DNA expression unit to replace the replaceable gene sequence, the resulting vector contains the promoter for the second selection marker gene, thereby enabling the expression of the second selection marker. This allows for the screening of vectors that have not successfully inserted into a specific site of the basic DNA expression unit, resulting in vectors that have successfully inserted the target gene or the foreign gene into a specific site of the basic DNA expression unit. Therefore, by inserting the aforementioned basic DNA expression unit into the reserved insertion site in the cell, and then inserting the target gene DNA expression unit into the specific site of the basic DNA expression unit through gene editing, it is possible to obtain stable cell lines with targeted insertion with a higher probability, and reduce the screening operations after the insertion of the target gene, greatly shortening the screening time after the insertion of the target gene, and quickly realizing the production and research applications related to the target gene.

[0046] The FRT3 and FRT gene sequences in the basic DNA expression unit serve as recognition sites for the recombinase FLP, enabling the replacement of the sequence between FRT3 and FRT in the Master pad with the sequence between FRT3 and FRT in the target gene DNA expression unit.

[0047] The basic DNA expression unit, also known as the master pad, is designed to insert itself into the genome at a specific site, reserving insertion sites for the target gene and selection markers. Furthermore, its own selection markers (first selection markers) must be distinct from the target gene's selection markers (second selection markers). Please refer to [link to relevant documentation]. Figure 1 The first promoter can initiate the expression of the first selection marker, thereby selecting and removing cells that have failed to successfully insert the basic DNA expression unit. Along the 5' to 3' direction, in the master pad without the inserted target gene DNA expression unit, the first promoter can initiate the expression of the first selection marker, and the first terminator is before the second selection marker gene sequence, therefore the second selection marker cannot be expressed. However, in the master pad with the inserted target gene DNA expression unit, the first promoter can initiate the expression of the target gene, and the terminator of the target gene and the promoter of the second selection marker are between FRT3 and FRT, enabling not only the expression of the target gene but also the expression of the second selection marker, thereby selecting and removing cells that have failed to successfully insert the target gene DNA expression unit. It should be noted that the cell line to which the basic DNA expression unit is to be inserted does not contain the second selection marker gene, thus enabling single-copy site-specific insertion of the target gene DNA expression unit.

[0048] The target gene sequence (MCS) is a multiple cloning site, which can be used to construct exogenous genes into the target gene DNA vector through double enzyme digestion or homologous recombination according to actual needs.

[0049] In practice, the basic architecture DNA expression unit (Masterpad) can be synthesized according to the above-described structure. This Masterpad is then constructed on the pUC19 plasmid for replication and amplification. A large number of linearized fragments, i.e., the basic architecture DNA vector, are obtained through plasmid digestion. This basic architecture DNA vector is then mixed with an sgRNA plasmid and electroporated into cells. Gene insertion is performed using a CRISPR system. Cell lines successfully inserted with the basic architecture DNA expression unit are selected using a first selection marker. These cell lines can then be used for subsequent site-specific insertion of various target genes. The target gene DNA vector serves as the vector for the target gene. The efficient FLP / FRT gene editing method enables site-specific insertion of exogenous or target genes. Cell lines with inserted target gene DNA expression units are then screened using a second selection marker. Only cells where the target gene is inserted at a predetermined location on the Masterpad will initiate expression of the second selection marker gene, thus passing the second selection marker-related screening. This technical solution saves a significant amount of work and time in single-clone screening and gene insertion site verification. Based on cell lines modified with the basic architecture DNA vector, target genes or exogenous genes can be inserted at specific sites, facilitating the application and promotion of stable transgenic cells.

[0050] The first and second screening marker gene sequences are each selected from either the hygromycin gene sequence or the glutamine synthase gene sequence. It should be noted that when the first screening marker gene sequence is the hygromycin gene sequence, the second screening marker gene sequence is the glutamine synthase gene sequence; conversely, when the first screening marker gene sequence is the glutamine synthase gene sequence, the second screening marker gene sequence is the hygromycin gene sequence.

[0051] Furthermore, the first selection marker gene sequence is the hygromycin (HygR) gene sequence. The first selection marker gene sequence is not limited to the hygromycin gene sequence; other selection marker gene sequences such as the glutamine synthase gene sequence or the puromycin gene sequence can also be used, as long as they can screen cell lines inserted into the Master pad. It should be noted that the first selection marker cannot be the same as the second selection marker. For example, when hygromycin is selected as the first selection marker, it cannot be selected as the second selection marker. When the first selection marker gene sequence is the hygromycin gene sequence, hygromycin is added to the selection medium to screen cell lines inserted into the Master pad; the surviving cell lines are those with the Master pad inserted.

[0052] The second selection marker gene sequence is the glutamine synthase (GS) gene sequence. The second selection marker gene sequence is not limited to the glutamine synthase gene sequence; other selection marker gene sequences such as hygromycin or puromycin gene sequences can also be used, as long as they can screen cell lines after the insertion of the target gene DNA expression unit. It should be noted that the second selection marker cannot be the same as the first selection marker. For example, when glutamine synthase is selected as the second selection marker, it cannot be selected as the first selection marker. When the second selection marker gene sequence is the glutamine synthase gene sequence, only cells where the target gene has been inserted at the predetermined position in the Master pad will initiate the expression of the glutamine synthase gene, thus enabling them to survive in L-glutamine-free culture media.

[0053] The first promoter gene sequence and the second promoter gene sequence are each selected from either a CMV promoter gene sequence or an SV40 promoter gene sequence. Further, the first promoter gene sequence is a CMV promoter gene sequence. That is, the first promoter is a CMV promoter. Alternatively, the first promoter can also be an SV40 promoter or a chicken β-actin promoter, etc. Using a CMV promoter provides strong transcriptional activity and efficiently drives gene expression. The second promoter gene sequence is an SV40 promoter gene sequence. That is, the second promoter is an SV40 promoter. Alternatively, the second promoter can also be a CMV promoter or a chicken β-actin promoter, etc. Using an SV40 promoter has strong initiation ability and can efficiently drive gene expression.

[0054] The first terminator gene sequence and the second terminator gene sequence are each selected from either the β-globin polyA gene sequence or the SV40 polyA gene sequence. Further, the first terminator gene sequence is the β-globin polyA gene sequence. That is, the first terminator is β-globin polyA. Alternatively, the first terminator can also be a gene sequence such as SV40 polyA that causes mRNA synthesis to stop at a specific position and begin degradation. The second terminator gene sequence is the SV40 polyA gene sequence. That is, the second terminator is SV40 polyA. Alternatively, the second terminator can also be a gene sequence such as β-globin polyA that causes mRNA synthesis to stop at a specific position and begin degradation.

[0055] It should be noted that the above conditions can be set separately or simultaneously. Preferably, when the first screening marker gene sequence is the hygromycin gene sequence, the second screening marker gene sequence is the glutamine synthase gene sequence, the first promoter gene sequence is the CMV promoter gene sequence, the second promoter gene sequence is the SV40 promoter gene sequence, the first terminator gene sequence is the β-globin polyA gene sequence, and the second terminator gene sequence is the SV40 polyA gene sequence, better gene editing and screening effects can be obtained.

[0056] Furthermore, the first promoter gene sequence is a CMV promoter gene sequence, and the basic DNA expression unit further includes a CMV enhancer gene sequence, which is located between the 5' homology sequence and the first promoter gene sequence. The CMV enhancer can effectively drive the CMV promoter, thereby increasing gene expression levels; therefore, the combined use of the CMV enhancer and the CMV promoter can improve gene expression efficiency.

[0057] The basic DNA expression unit also includes an intron A gene sequence (Intron A) and an intron B gene sequence (Intron B). The intron A gene sequence is located between the first promoter gene sequence and FRT3, and the intron B gene sequence is located between FRT3 and the second selectable marker gene sequence. Adding the intron A and intron B gene sequences can promote gene expression.

[0058] Furthermore, the basic architecture DNA vector is obtained by modifying the pUC19 plasmid. The basic architecture DNA expression unit is constructed on the pUC19 plasmid for replication and amplification. A large number of linearized fragments are obtained by plasmid enzyme digestion. The linearized fragments are the basic architecture DNA vector, which is mixed with the sgRNA plasmid and electroporated into cells. Gene insertion can be completed by the CRISPR system.

[0059] The target gene DNA vector was obtained by modifying the pUC19 plasmid. The target gene DNA vector was constructed by inserting the target gene DNA expression unit into the pUC19 plasmid. Depending on the specific needs, the exogenous gene can be constructed into a vector with [specific expression characteristics] through double enzyme digestion or homologous recombination. Figure 2 The target gene DNA vector (pDDCR plasmid) with the structure shown is specifically positioned to replace the target gene sequence, thereby enabling the site-specific insertion of exogenous genes through efficient FLP / FRT gene editing methods.

[0060] Furthermore, it should be noted that the 5' homology sequence and the 3' homology sequence are sequences within 300-1000 bp of the sgRNA. The 5' homology sequence and the 3' homology sequence within 300-1000 bp of the sgRNA are used to determine the insertion site of the Master pad. BamHI can be selected as the restriction endonuclease for Master pad linearization, while the pUC19 plasmid itself must not contain this restriction site.

[0061] Furthermore, the nucleotide sequence of the basic architecture DNA expression unit is shown in SEQ ID NO: 1, specifically as follows:

[0062]

[0063] The nucleotide sequence of the target gene DNA expression unit is shown in SEQ ID NO: 2, specifically:

[0064]

[0065] This invention further proposes a method for site-specific insertion of exogenous gene sequences, using the DNA vector described above. The method for site-specific insertion of exogenous gene sequences includes the following steps:

[0066] S10. Select the cell line and genomic site to be modified by targeted insertion, and design sgRNA according to the site and the gene editing protein used. Construct the sgRNA on the plasmid to obtain the sgRNA plasmid.

[0067] S20. Using sgRNA plasmid, the basic DNA expression unit is electroporated into the cell line to be modified, and intermediate recombinant cell line is obtained through first antibiotic selection.

[0068] S30. Using the FLP / FRT recombinase system, the target gene DNA expression unit with the target gene is inserted into the intermediate recombinant cell line, and the cell line with a site-directed single copy of the target gene is obtained through second resistance screening.

[0069] The cell line to be modified is a cell line that does not contain the second resistance selection gene.

[0070] In the technical solution provided in this application, cell lines are modified by pre-inserting a single copy of some elements into a specific site. Monoclonal cells are obtained through first-resistance screening. Subsequent insertion of the target gene is performed on the modified intermediate recombinant cell line. Since the second selection marker gene is pre-inserted into the genome, and its promoter is introduced during the insertion of the target gene, even if the target gene is mistakenly inserted into other sites, the cell line will still not pass the relevant screening of the second selection marker. Therefore, cell lines that pass the relevant screening of the second selection marker theoretically have the same genetic background, thereby significantly reducing the screening time when inserting the target gene and ensuring the achievement of site-specific single-copy insertion, which is conducive to the rapid realization of its production and research applications.

[0071] Furthermore, the second screening marker is glutamine synthase, and the cell line to be modified is Chinese hamster ovary cells with the glutamine synthase biselequé (GS) knocked out. Chinese hamster ovary cells with the glutamine synthase biselequé knocked out (CHO-K1 2H11) cannot survive in L-glutamine-free medium. CHO-K12H11 (2H11-MP) with the inserted basic DNA expression unit cannot survive in L-glutamine-free medium because the GS gene cannot be expressed; only 2H11-MP with the inserted target gene DNA expression unit can initiate GS gene expression and therefore can survive in L-glutamine-free medium. Therefore, using L-glutamine-free medium allows for the screening of cell lines with site-specific single-copy insertion of the target gene.

[0072] In step S10, the CHO-K1 2F10 (CGMCC 45325) cell line was obtained by domestication of CHO-K1 (ECACC), and the GS biallelic gene was knocked out to obtain the CHO-K1 2H11 cell line (also referred to as 2H11 or CHO-K1 2H11 cell line in other positions in this application). Chromosome walking technology was used to sequence and identify loci in the CHO-K1 2H11 cell line genome suitable for site-specific insertion of exogenous genes. These loci should be stable, highly expressed, and not affect the expression of other genes. Preferably, the locus suitable for site-specific insertion of exogenous genes is LOC103161380. Using CRISPR-Cas9 technology, the three pairs of sgRNAs with the highest comprehensive scores—namely sgRNA1, sgRNA2, and sgRNA3—were selected from the locus sequence using the CRISPOR website (http: / / crispor.tefor.net / ).

[0073] The sgRNA1 includes sgRNA1-F and sgRNA1-R, and the gene sequence of sgRNA1-F is shown in SEQ ID NO: 3, specifically as follows:

[0074] caccGTTGGTTCAAAAATCTTCAG

[0075] The gene sequence of the sgRNA1-R is shown in SEQ ID NO: 4, and is as follows:

[0076] aaacCTGAAGATTTTTGAACCAAC

[0077] The sgRNA2 includes sgRNA2-F and sgRNA2-R, and the gene sequence of sgRNA2-F is shown in SEQ ID NO: 5, specifically as follows:

[0078] caccGGTGGCTAAGGAGATGAACT

[0079] The gene sequence of the sgRNA2-R is shown in SEQ ID NO: 6, specifically:

[0080] aaacAGTTCATCTCCTTAGCCACC

[0081] The sgRNA3 includes sgRNA3-F and sgRNA3-R, and the gene sequence of sgRNA3-F is shown in SEQ ID NO: 7, specifically as follows:

[0082] caccGCCTTGCACCATGTGGGTTTC

[0083] The gene sequence of the sgRNA3-R is shown in SEQ ID NO: 8, and is as follows:

[0084] aaacGAAACCCACATGGTGCAAGGC

[0085] In step S20, the sgRNA is one of sgRNA1, sgRNA2, and sgRNA3. The basic DNA vector is inserted into the cell line to be modified using the sgRNA. In some embodiments, the CRISPR-Cas9 gene editing method is used to guide the insertion of the basic DNA template into the cell line to be modified at a specific site to complete the modification of the cell line.

[0086] The insertion of the basic DNA expression unit into the genome is determined through a first resistance selection process. This first resistance selection is based on the sequence of a first selection marker gene; for example, when the first selection marker gene is the hygromycin gene, the first resistance selection involves transferring cells to a medium containing hygromycin for culture. Genome sequencing is used to determine whether the basic DNA expression unit is correctly inserted at the locus. Simultaneous electroporation of red and green fluorescent genes, followed by flow cytometry to determine the red-green light ratio, confirms single-copy insertion of the basic DNA expression unit. Transfection of the red and green fluorescent genes using FLP / FRT gene editing methods further confirms single-copy insertion. Through these selections, intermediate recombinant cell lines are obtained, ready for subsequent insertion of various target genes or exogenous genes.

[0087] Before step S30, the method further includes inserting the target gene or a foreign gene into the target gene DNA expression unit. For example, when the foreign gene is an Anti-HCG antibody gene, the Anti-HCG antibody sequence is constructed onto the pDDCR plasmid to replace the target gene sequence, thereby obtaining a target gene DNA expression unit constructed with the Anti-HCG antibody gene, and the vector is the pDDCR plasmid.

[0088] In step S30, the target gene DNA expression unit containing the target gene is inserted into the intermediate recombinant cell line, and a cell line with a site-specific single copy of the target gene is obtained through a second resistance screening.

[0089] The method for site-specific insertion of exogenous gene sequences provided by this invention can be used to achieve stable and efficient expression of exogenous genes with a clear genetic background, significantly shorten the construction time of stable cell lines, and promote the large-scale production and application of cell-expressed antibodies or proteins.

[0090] The present invention also proposes the application of the DNA vector described above in the study of recombinant protein / antibody expression or gene function.

[0091] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0092] Example 1: Selection of a site-specific insertion locus

[0093] 1. Performance validation of the MM16-14B10 cell line

[0094] In preliminary experiments, it was found that the stable MM16-14B10 cell line expressed up to 3 g / L of MM16 antibody. Subsequent investigations into its growth performance revealed that this stable cell line, in Quacell CHO CD04 medium, exhibited good cell homogeneity (>90%) and a stable viability of over 98% after 20 passages, with an average doubling time of 22 hours, demonstrating excellent passage stability. (The text then abruptly shifts to a different topic: "At 0.5 × 10...") 6 Cells / ml were seeded, and the counting density was 4 × 10⁶ cells / ml every 3 days. 6 ~6×10 6 cells / ml; at 0.3×10 6 Cells / ml were seeded at a density of 6.3 × 10⁻⁶ every 4 days. 6 ~7.5×10 6 The cell density showed a slow overall increase, consistent with the growth pattern of CHO cells. P0, P5, P10, P15, and P20 passages were cultured in shake flasks using Fed batches, and antibody yield and quality were monitored. Antibody expression levels were acceptable, and quality remained relatively stable, indicating good stability of the target gene expression. Simultaneously, the genome of P5, P10, P15, and P20 passages of M16-14B10 cells was extracted. Using the P0 passage as a template, qPCR was performed using the optimal primers for the target gene and the internal reference gene B2M. The copy number deviations of P5, P10, P15, and P20 relative to P0 were 1%, 0%, 5%, and 11%, respectively, indicating relatively stable genetic material and good genetic stability. Further analysis revealed that the MM16-14B10 cell line is a single-copy antibody. In conclusion, the insertion site of the MM16 antibody exhibits good performance and can be used as a site for single-copy targeted insertion.

[0095] 2. Determine the location of the MM16 antibody insertion locus.

[0096] The unknown sequence region was amplified using chromosome walking technology. Three specific primers, 3'-SP1, 3'-SP2, and 3'-SP3, were designed for the 3' end of the known sequence, with nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. Three specific primers, 5'-SP1, 5'-SP2, and 5'-SP3, were designed for the 5' end, with nucleotide sequences shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0097] The nucleotide sequence of 3'-SP1 is as follows: GCTCCGTGGTGCACGAGGGCCTGCAC

[0098] The nucleotide sequence of 3'-SP2 is as follows: GAGATCCCTCGAGATCCATTGTGCT

[0099] The nucleotide sequence of 3'-SP3 is as follows: CATGCGTCATTTTGACTCACGCGGTC

[0100] The nucleotide sequence of 5'-SP1 is as follows: AGCCTCTCACTACTTCTGGAATAGC

[0101] The nucleotide sequence of 5'-SP2 is as follows: GCCTGGGGACTTTCCACACCCTAACTG

[0102] The nucleotide sequence of 5'-SP3 is as follows: GCTGTCCCTCATAAAAGTTTTGTTAC

[0103] The design principles of the specific primers were followed according to the instructions of the Takara Genome Walking Kit (Code NO. 6108). Three rounds of PCR were performed using three specific primers at the 3' and 5' ends, and three degenerate primers provided with the kit. The template for each subsequent round was the PCR product from the previous round. The PCR products from all three rounds were subjected to agarose gel electrophoresis. The gel electrophoresis results are shown below. Figure 4 As shown. Clear electrophoretic bands from the third round of PCR products were recovered by gel excision. After TA cloning of the PCR products from both ends, 10 strains were randomly selected for sequencing. Sequencing results showed that the 10 sequencing fragments at the 5' end and the 10 sequencing fragments at the 3' end were the same sequence. Alignment analysis of the sequencing results with the CHO-K1 whole genome confirmed that the locus inserted in the MM16-14B10 cell line was LOC103161380.

[0104] Example 2: Design, Synthesis, and Validation of sgRNA

[0105] 1. Design and synthesis of sgRNA

[0106] Using CRISPR-Cas9 technology, the three pairs of sgRNAs with the highest overall scores were selected from the CRISPOR website (http: / / crispor.tefor.net / ) based on the locus sequence. These pairs were named sgRNA1, sgRNA2, and sgRNA3, respectively. sgRNA1 includes sgRNA1-F and sgRNA1-R; sgRNA2 includes sgRNA2-F and sgRNA2-R; and sgRNA3 includes sgRNA3-F and sgRNA3-R. The gene sequences of sgRNA1-F are shown in SEQ ID NO: 3; sgRNA1-R is shown in SEQ ID NO: 4; sgRNA2-F is shown in SEQ ID NO: 5; sgRNA2-R is shown in SEQ ID NO: 6; sgRNA3-F is shown in SEQ ID NO: 7; and sgRNA3-R is shown in SEQ ID NO: 8. As shown in IDNO:8, the sample was synthesized by Shanghai Jereh Biotechnology Co., Ltd. and constructed onto the PX458 plasmid. This plasmid contains the functional elements required for gene editing, including gRNA scaffold, Cas9 protein gene sequence, and green fluorescent protein GFP.

[0107] 2. Verification of sgRNA editing efficiency

[0108] Preparation of CHO-K1 2H11 transfected cells: This invention is based on the CHO-K12F10 (CGMCC 45325) cell line obtained by domestication of CHO-K1 (ECACC), and then the CHO-K1 2H11 cell line (hereinafter referred to as 2H11) obtained by knocking out the GS biallelic gene. This cell line requires 4-6 mM L-glutamine supplementation in the culture medium to maintain normal growth. One flask of 2H11 cells was seeded, and the cell density was controlled at 5 × 10⁶ cells / mL. 5 Cells / mL were incubated at 180 rpm for 24 h in a humidified orbital vibrating incubator at 37°C with 5% CO2. Cells were counted using an automated cell counter via the trypan blue rejection method to calculate the appropriate volume of culture required for transfection (cell density adjusted to 1 × 10⁻⁶ cells / mL). 7 Transfer the cells (cells / mL) to a sterile conical centrifuge tube, centrifuge at 1000 rpm for 5 minutes at 15–25°C, carefully aspirate the supernatant, and resuspend the cell pellet in PBS (pH 7.4) for later use.

[0109] Transfection of sgRNA plasmid: Prepare complete culture medium (with 6 mM L-glutamine added) as transfection and recovery medium, and label 3 T-25cm cells. 2Under aseptic conditions, 4.0 mL of complete culture medium was added to each suspension cell culture flask. Then, 0.8 mL of cell suspension and 30 μg of sgRNA1, sgRNA2, or sgRNA3 plasmids were gently mixed in three sterile microcentrifuge tubes, respectively. The mixture was then transferred to three cooled electroporation cuvettes and electroporated using specific parameters (300 V voltage, 950 μF capacitance, exponentially decaying pulse). After electroporation, the cells were transferred to a T-25 cm⁻¹ container containing 4.0 mL of complete culture medium. 2 The cells were incubated in a suspension cell culture flask at 37°C and 5% CO2 for 48 hours.

[0110] Editing efficiency verification: Two days after transfection, genomic DNA was extracted from cells and PCR amplification was performed using verification primers. The verification primers (YZ primer) included YZ primer-F and YZ primer-R. The sequence of YZ primer-F is shown in SEQ ID NO:15, specifically:

[0111] TGGAGTGTTCTACATGGCAC

[0112] The sequence of YZ primer-R is shown in SEQ ID NO:16, specifically:

[0113] TCTGTCTGAGGTGGAGGCTC

[0114] Half of the PCR products were treated with T7 Endonuclease I, and the other half were not. Both were analyzed by gel electrophoresis to determine the bands. The results are as follows: Figure 5 As shown in the figure, if the product with added T7 Endonuclease I shows multiple bands, while the product without added T7 Endonuclease I shows a single band, then the sgRNA can cleave the gene. For the cleavage principle and enzyme digestion system of T7 Endonuclease I, please refer to the Novizan T7 Endonuclease I (EN303) product manual. As can be seen from the figure, all three pairs of sgRNAs can perform gene editing; ultimately, sgRNA2 was selected for subsequent CRISPR gene editing.

[0115] Example 3: Design and synthesis of Master pad and pDDCR plasmid

[0116] 1. Design and synthesis of master pad sequences

[0117] The Master pad is a sequence pre-integrated into the CHO-K1 2H11 cell genome, synthesized by General Biotech Inc., and constructed onto the pUC19 plasmid for replication and amplification. A large number of linearized fragments are obtained through plasmid digestion, mixed with the sgRNA2 plasmid, and electroporated into cells. Gene insertion is then completed using the CRISPR system. A schematic diagram of the Master pad structure is shown below. Figure 1 As shown, the nucleotide sequence of the Master pad is shown in SEQ ID NO:1.

[0118] BamHI was chosen as the restriction endonuclease for Master pad linearization, requiring that the pUC19 plasmid itself does not contain this restriction site.

[0119] The sequence within 300-1000 bp of the endonuclease sgRNA2 was used as the 5' and 3' homologous arms to determine the insertion site of the master pad. The nucleotide sequence of the 5' homologous arm is shown in SEQ ID NO:17, specifically:

[0120] GGAGACGGGGTTTCTCTGGGGGGTTGGAGCCTGTCCTGGCCCTCACTCTGTAGACCAGGCTAGCCTCGAACTCAGAGAGATCCGCCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCGTGTGCCACCAACGCCCAGCTCCCCCTGGAGTGTTCTACATGGCACAGACCATCCAAATCTTGACTGGATTCTCTGCCTCTAAATTTGAGCACTTTCTAAATTTATTCTTTCTTTTTTTGAGACA AGGTCTCACGTGTCCTACTCTGCCCCGTCTCCCTGTGTAGTCAAGGATAATCTTGAACTCCTGATCCTCCTGCCTCCACTTCTAAGTGCTGAGAACAAGGCATGTCCCACTGTGCCTGGCTCTGAGTTACTGACCAAATGGACAAATAATCATTTCATTAGTTGGTTCAAAAATCTTCAGTGGGGTTGGCCAGATGGCTTAGCAGATGAAGGCACTTGCTGTCCAGCCTCATGACCAGAGTTTGAT

[0121] The nucleotide sequence of the 3' homologous arm is shown in SEQ ID NO:18, specifically:

[0122] AGGGACACATTCTAAGACATGTCAGGTGCCCGGAGCTCCACCTCAGACAGAGCCCTCTGTACTAATTTTTTTTTTTCCTATGTGCTTGCAGCAT TGCTACACAGACCATGATTAATCTGTCCTTACATGATTTAAGTCCTGCTGTCTACCCCTTTACTACTCTTGGGCTTTTGAGTCATTACTAAGTAAAATAAGGATTATTGAACAGCAGCATGTGGAACCCGGGCAGTAAATCTGACAAGCAAGGGAGGTACCAAGTGAGTAATTCCATGGCTACTATGTGGCTAATGACT CTACTAACTGGTAGACATGCTGGACAGGATGATTCATGTTCTGGTAGGGACAGGGTGAGATGCTGTGTGGCTCAGAGCACTGTTCAACCTGAAGCTCATGTCTTGAACATTTCTGGAATTTTCCATGTCATATTTCTAAACTGTGGTTGGCTGGGTAAGTGAAAGTATAGGAAATAAAGCCACAGATAAGTGGGGACCAC

[0123] Guide RNA was used to initiate hygromycin (HygR) gene expression using the CMV strong promoter, serving as a selection marker for Master pad insertion;

[0124] β-globin polyA serves as a terminator for hygromycin (HygR) expression;

[0125] The FRT3 and FRT gene sequences are reserved as sites for recognition by the recombinase FLP;

[0126] Reserve the glutamine synthase gene (GS) as a selection marker for subsequent insertion of the target gene;

[0127] SV40 polyA is used as the terminator for GS.

[0128] 2. Design and synthesis of pDDCR plasmid

[0129] pDDCR is a plasmid that matches the 2H11-MP cell line for site-directed insertion of foreign genes, and it is derived from the pUC19 plasmid. It contains the following components, as shown in the schematic diagram below. Figure 2 :

[0130] The FRT3 and FRT gene sequences are the sites recognized by the recombinase FLP. The sequence between the two can completely replace the sequence between FRT3 and FRT on the Master pad in a "copy and paste" manner. MCS is a multiple cloning site, which can construct foreign genes into pDDCR plasmids through double enzyme digestion or homologous recombination as needed. β-globin polyA serves as a terminator for the target gene. SV40 promoter can initiate the expression of the GS gene and serves as a selection marker to determine whether the target gene has been correctly inserted.

[0131] Example 4: Master pad-based insertion of CHO-K1 2H11 cells and verification

[0132] 1. Insert 2H11 cells into the Master pad.

[0133] Linearization of Master pad: The Master pad-pUC19 plasmid was transformed into DH5α, cultured overnight in LB medium, and the plasmid was extracted and linearized Master pad was obtained by single digestion with BamHI restriction endonuclease.

[0134] Preparation of 2H11 transfected cells: The preparation process for 2H11 cells is the same as in Example 2, except that 4-6 mM L-glutamine needs to be added to the culture medium.

[0135] Transfection of linearized Masterpad and sgRNA2 plasmid: Prepare complete culture medium (with 6 mM L-Gln added) as transfection and recovery medium, and label T-25cm 2 Under aseptic conditions, add 4.0 mL of complete culture medium to the suspension cell culture flask. Gently mix 0.8 mL of cell suspension, 10 μg of sgRNA2 plasmid, and 30 μg of linearized Masterpad in a sterile microcentrifuge tube. Transfer the mixture to a cooled electroporation cuvette and perform electroporation according to specific parameters (300 V voltage, 950 μF capacitance, exponentially decaying pulse). After electroporation, transfer the cells to a T-25 cm⁻¹ container containing 4.0 mL of complete culture medium. 2 The cells were incubated in a suspension cell culture flask at 37°C and 5% CO2 for 48 hours.

[0136] 2. Monoclonal screening

[0137] 48 hours after transfection, cells were transferred from T-25cm 2Aseptically transfer the culture flask to a 15ml sterile conical tube, centrifuge at 1000rpm for 5 minutes at 15-25℃, and carefully remove the supernatant. Resuspend the cell pellet in 10.0mL selection medium (CHO fusion medium containing 400μg / mL hygromycin), aseptically aspirate a small amount of sample, and count the cells using an automated cell counter via trypan blue exclusion assay. Perform the cell count at a rate of 1–2 × 10⁻⁶ cells / mL. 4 Calculate the total volume required to achieve the desired cell / well density. Dilute the cells to the required density using an appropriate amount of selection medium. Use a multichannel pipette to add the diluted cells to a 96-well plate (200 μl per well). Place the cells in a humidified CO2 incubator at 37°C. After 5 days, remove the plates to observe cell growth; do not remove them during this period to avoid interfering with recovery. Replace the evaporated medium weekly with selection medium. Once the cells reach approximately 80% confluence and appear healthy under a microscope, allocate cells at a 1:4 dilution to 96-well plates containing 200 μl of selection medium (first passage). This should be done between days 10 and 21 post-inoculation. After multiple rounds of passage, 15 single clones were finally selected and named 2H11-MP-1, 2H11-MP-2, 2H11-MP-3, 2H11-MP-4, 2H11-MP-5, 2H11-MP-6, 2H11-MP-7, 2H11-MP-8, 2H11-MP-9, 2H11-MP-10, 2H11-MP-11, 2H11-MP-12, 2H11-MP-13, 2H11-MP-14, and 2H11-MP-15. All of them were used for library construction and cryopreservation.

[0138] 3. Sequencing to determine the insertion status of the master pad.

[0139] The whole genomes of 15 monoclonal clones were extracted and amplified by PCR using sequencing primers. The sequencing primers included CX primer-F and CX primer-R. The sequence of CX primer-F is shown in SEQ ID NO:19, and is as follows:

[0140] CACCCATCCCTTCCTTTATGT

[0141] The sequence of CX primer-R is shown in SEQ ID NO:20, specifically:

[0142] CCCTGTCCCTACCAGAACATG

[0143] The PCR products and primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to determine whether the Masterpad was correctly inserted into the selected locus. According to the sequencing results, all 15 monoclonal clones were correctly inserted into the selected locus.

[0144] 4. Use the FLP / FRT gene editing method to transfect the red and green fluorescent genes to determine whether the master pad is a single copy.

[0145] Preparation of pDDCR-DsRED, pDDCR-eGFP, and pGAP-FLP plasmids: The red and green fluorescent genes were constructed into the pDDCR plasmid through two restriction enzyme sites, BstBI and SalI, to obtain pDDCR-DsRED and pDDCR-eGFP. Plasmid construction was performed by General Biotechnology Co., Ltd. The pDDCR-DsRED and pDDCR-eGFP plasmids were transformed into DH5α, cultured overnight in LB medium, and then the plasmids were extracted for later use. The pGAP-FLP plasmid was purchased from Hangzhou Hongsai Biotechnology Co., Ltd. This plasmid was transformed into DH5α, cultured overnight in LB medium, and then the plasmid was extracted for later use.

[0146] Preparation of 2H11-MP transfected cells: The preparation process of 2H11-MP is the same as in Example 2. 4-6 ml of glutamine needs to be added to the culture medium. Transfected cells need to be prepared for 15 single clones.

[0147] Transfection of pDDCR-DsRED, pDDCR-eGFP, and pGAP-FLP plasmids: Medium containing 6 mM L-glutamine was used as the transfection and recovery medium, labeled at T-25cm. 2 Under aseptic conditions, add 4.0 mL of complete culture medium to the suspension cell culture flask. Gently mix 0.8 mL of cell suspension, 5 μg of pGAP-FLP plasmid, 15 μg of pDDCR-DsRED plasmid, and 15 μg of pDDCR-eGFP plasmid in a sterile microcentrifuge tube. Transfer the mixture to a cooled electroporation cuvette and perform electroporation according to specific parameters (300 V voltage, 950 μF capacitance, exponentially decaying pulse). After electroporation, transfer the cells to a T-25 cm⁻¹ container containing 4.0 mL of complete culture medium. 2 The cells were incubated in a suspension cell culture flask at 37°C and 5% CO2 for 48 hours.

[0148] Passaging of transfected cells: 48 hours after transfection, T-25cm cells were passaged... 2 Centrifuge the cells in the culture flask at 1000 rpm for 5 minutes at 15-25℃, carefully remove the supernatant, and resuspend the cell pellet in 10.0 mL of GS selection medium (without L-glutamine). Then, divide the resuspended cells into 1×10⁻⁶ cells. 6 The total number of cells at T-75cm 2 The cells were expanded in culture flasks and incubated statically at 37°C and 5% CO2 for 12 days. After 12 days, cell density was measured, and cells were injected weekly at a rate of 3 × 10⁶ cells / week. 5Viable cells / mL were passaged twice.

[0149] Flow cytometry was used to determine the red-green fluorescence ratio: Cells after two passages were transferred to 50 mL centrifuge tubes, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cell pellet was sent to the Scientific Compass testing platform for flow cytometry analysis to determine the red-green fluorescence ratio. According to the analysis results, in 14 out of 15 single clones, cells emitting only red or green light accounted for more than 99% of the total cell count, and the red:green ratio was approximately 1:1, indicating that the Masterpad in all 14 single clones was a single copy insertion. Based on a comprehensive comparison of cell line growth status, the final cell lines 2H11-MP-3, 2H11-MP-8, and 2H11-MP-12 were selected for subsequent site-specific insertion.

[0150] Example 5: Site-directed insertion of the Anti-HCG antibody gene into 2H11-MP cells and identification.

[0151] To verify the effectiveness of targeted insertion into cell lines for antibody expression, this embodiment uses the Anti-HCG antibody sequence for targeted insertion into the 2H11-MP-3 cell line for evaluation.

[0152] 1. Anti-HCG antibody sequence was inserted into 2H11-MP-3 cells at a specific site.

[0153] Anti-HCG antibody sequences were constructed into the pDDCR plasmid: The heavy and light chain sequences of the anti-HCG antibody were ligated using IRES, and then constructed into the pDDCR plasmid via BstBI and SalI restriction sites. Plasmid construction was performed by General Biotech Inc. The pDDCR-Anti-HCG plasmid was transformed into DH5α, cultured overnight in LB medium, and then the plasmid was extracted for later use. The preparation of the pGAP-FLP plasmid was the same as in Example 4.

[0154] Preparation of 2H11-MP-3 transfected cells: The preparation process of 2H11-MP-3 is the same as in Example 2, except that 4-6 mM L-glutamine needs to be added to the culture medium.

[0155] pDDCR-Anti-HCG transfection into 2H11-MP-3: Medium containing 6 mM L-glutamine was used as the transfection and recovery medium, labeled T-25cm. 2Under aseptic conditions, add 4.0 mL of complete culture medium to the suspension cell culture flask. Gently mix 0.8 mL of cell suspension, 5 μg of pGAP-FLP plasmid, and 25 μg of pDDCR-Anti-HCG plasmid in a sterile microcentrifuge tube. Transfer the mixture to a cooled electroporation cuvette and perform electroporation according to specific parameters (300 V voltage, 950 μF capacitance, exponentially decaying pulse). After electroporation, transfer the cells to a T-25 cm⁻¹ container containing 4.0 mL of complete culture medium. 2 The cells were incubated in a suspension cell culture flask at 37°C and 5% CO2 for 48 hours.

[0156] Passaging and fermentation of 2H11-Anti-HCG cells: Cells were passaged after transfection as in Example 4. After two passages, cells were fermented at a rate of 3 × 10⁻⁶ cells / year. 5 The inoculum concentration was set at the specified level, and fed-batch fermentation was carried out in 125 mL shake flasks. L-glutamine was not required in the culture medium. Feeding began on day three, with 4% Feed A and 0.4% Feed B added every other day at an initial fermentation volume of 30 mL. From day five onwards, glucose was added every other day until a concentration of 5 g / L was reached. The survival rate upon sample collection must be >70%, and the maximum culture period is 13 days.

[0157] 2. Anti-HCG antibody yield assay

[0158] To determine whether the site-directed insertion cell line efficiently expresses Anti-HCG antibody, antibody expression yield was measured using a biochemical analyzer.

[0159] The experimental procedure is as follows:

[0160] Sample pretreatment: Take 1 mL of the collected fermentation broth, centrifuge at 10000 rpm for 2 minutes at 15-25℃ to remove the precipitate, and collect the supernatant as the sample to be tested;

[0161] Reagent preparation: Reagent R1, Reagent R2, 1×PBS (pH 7.4), goat anti-mouse secondary antibody, calibrators (concentrations of 1000 μg / mL, 500 μg / mL, 200 μg / mL, 50 μg / mL, 10 μg / mL, and 0 μg / mL), and quality control samples (concentrations of 1000 μg / mL, 200 μg / mL, and 50 μg / mL).

[0162] Refer to the instruction manual for the Zhongyuan Huiji EXC800 biochemical analyzer to set the parameters and place the reagents, calibration, quality control, and samples into the corresponding trays;

[0163] Start calibration and quality control. If the deviation between the quality control value and the target value is within 20%, start testing the sample to be tested and repeat the test three times.

[0164] The test results showed that the three test results were 1201 mg / L, 1210 mg / L, and 1197 mg / L, with a test deviation of 3.29%, indicating that the results were reliable. Finally, the fermentation yield of the 2H11-Anti-HCG cell line was determined to be approximately 1203 mg / L.

[0165] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A DNA vector for site-directed insertion of a foreign gene sequence, characterized in that, The DNA vector includes: A basic DNA vector includes a basic DNA expression unit, wherein the basic DNA expression unit comprises, from 5' to 3', a 5' homology sequence, a first promoter gene sequence, a replaceable gene sequence, a second selectable marker gene sequence, a second terminator gene sequence, and a 3' homology sequence. The basic DNA expression unit does not contain a promoter for the second selection marker gene. The marker can only be activated and expressed after the target gene DNA expression unit is correctly inserted into a specific site in the basic DNA expression unit, thereby selecting stable cell lines with site-directed insertion. The target gene DNA vector includes a target gene DNA expression unit, wherein the target gene DNA expression unit includes FRT3, the target gene sequence, the first terminator gene sequence, the second promoter gene sequence, and FRT in sequence from 5' to 3'. The replaceable gene sequence includes FRT3, a first screening marker gene sequence, a first terminator gene sequence, and FRT in sequence from 5' to 3', and the replaceable gene sequence can be replaced with the target gene DNA expression unit. The first screening marker gene sequence and the second screening marker gene sequence are respectively selected from either the hygromycin gene sequence or the glutamine synthase gene sequence; The first promoter gene sequence is a CMV promoter gene sequence, and the basic DNA expression unit further includes a CMV enhancer gene sequence, which is located between the 5' homology sequence and the first promoter gene sequence.

2. The DNA vector as described in claim 1, characterized in that, The first promoter gene sequence and the second promoter gene sequence are each selected from one of the CMV promoter gene sequence or the SV40 promoter gene sequence; and / or, The first terminator gene sequence and the second terminator gene sequence are respectively selected from either the β-globin polyA gene sequence or the SV40 polyA gene sequence.

3. The DNA vector as described in claim 2, characterized in that, The basic DNA expression unit further includes an intron A gene sequence and an intron B gene sequence, wherein the intron A gene sequence is located between the first promoter gene sequence and FRT3, and the intron B gene sequence is located between FRT3 and the first selection marker gene sequence.

4. The DNA vector as described in claim 1, characterized in that, The basic DNA vector was obtained by modifying the pUC19 plasmid. The basic DNA expression unit was constructed on the pUC19 plasmid for replication and amplification. A large number of linearized fragments were obtained by plasmid enzyme digestion to obtain the basic DNA vector. And / or, The target gene DNA vector is obtained by modifying the pUC19 plasmid. The target gene DNA vector is constructed by inserting the target gene DNA expression unit into the pUC19 plasmid.

5. The DNA vector as described in claim 1, characterized in that, The nucleotide sequence of the basic architecture DNA expression unit is shown in SEQ ID NO: 1; and / or, The nucleotide sequence of the target gene DNA expression unit is shown in SEQ ID NO:

2.

6. The use of a DNA vector as described in any one of claims 1 to 5 in recombinant protein expression or gene function research, wherein the use is for non-disease diagnosis and treatment purposes.

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