Method and system for site-specific integration of exogenous genes

Abstract

The invention relates to the technical field of biology, in particular to a method and system for site-specific integration of exogenous genes in a cell genome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and system for site-specific integration of foreign genes into the CHO cell genome. Background Art

[0002] Traditional integration of foreign genes into CHO cells is achieved through random integration. Usually, DNA fragments containing foreign genes are introduced into the interior of CHO cells by means of electroporation, chemical transformation, etc. Subsequently, the foreign genes will enter the nuclei of some CHO cells, and in the CHO cells into which the foreign genes enter the nuclei, some foreign genes will be integrated into certain sites of the CHO cell genome by unknown mechanisms. These integration sites are random, and this process is called random integration (RI).

[0003] The expression level and stability of foreign genes in CHO cells are related to the integration sites of the CHO cell genome, which is called the position effect of chromosomes. Since random integration has no obvious preference for the integration sites on the CHO cell genome, in cell line development (CLD) work, in order to screen cell lines with high yield, stability and adaptability to specific production processes that are integrated into good genomic sites, usually a large screening amount and a long screening period are required.

[0004] In order to improve the efficiency of CLD work and reduce the screening amount and period of CLD work, in recent years, different companies and research institutions have developed site-specific integration (SSI) processes. The site-specific integration process refers to using technologies such as integrases and transposons to integrate foreign genes into the expected sites of cell chromosomes. Compared with random integration, site-specific integration has the characteristics of simple molecular operation, high integration efficiency, high cell consistency, etc., and can significantly reduce the screening amount and period of CLD work and improve the efficiency of CLD work. The current bottleneck restricting the application of site-specific integration technology is the expression level of foreign genes. Compared with random integration, due to the small number of gene copies in single-site site-specific integration technology, the yield is difficult to exceed that of cell lines screened by random integration with high throughput.

[0005] Therefore, there is an urgent need in the art for a method that can achieve site-specific integration and at the same time improve the expression level of foreign genes. Summary of the Invention

[0006] Through in-depth research and exploration, the present inventor provides a method and related system for site-specific integration of foreign genes in the genome of CHO cells while simultaneously increasing the expression level of foreign genes. Specifically, the present invention is based on a unique integrase technology to integrate foreign genes into the genome of CHO cells. Surprisingly, this method has a high integration efficiency, good integration consistency among cells, and the expression level of foreign genes is significantly higher than that of the random integration control.

[0007] In some aspects, the present invention provides a method for site-specific integration of foreign genes in the genome of CHO cells, which includes introducing exogenous integrase-encoding nucleic acid and exogenous nucleic acid sequence into CHO cells, wherein the exogenous nucleic acid sequence contains a foreign gene and attB sequences or attP sequences located on both sides of the foreign gene, whereby the foreign gene is integrated into the genome of CHO cells under the action of the exogenous integrase. Preferably, the expression level of the foreign gene integrated thereby is significantly higher than that of the random integration control.

[0008] In some aspects, the present invention provides a CHO cell, which site-specifically integrates a foreign gene in the genome according to the method of the foregoing embodiments, wherein the foreign gene is site-specifically integrated into the genome of the CHO cell by relying on the exogenous integrase and the attB sequences or attP sequences located on both sides of the foreign gene. Preferably, the expression level of the foreign gene integrated thereby is significantly higher than that of the random integration control.

[0009] In some aspects, the present invention provides a system for site-specific integration of foreign genes in the genome of CHO cells, the system includes one or more vectors, wherein one or more vectors contain:

[0010] a. A foreign gene;

[0011] b. AttB sequences or attP sequences located on both sides of the foreign gene;

[0012] and / or c. Exogenous integrase-encoding nucleic acid, which can express exogenous integrase in CHO cells;

[0013] wherein a, b, and c can be located in the same or different vectors;

[0014] Thereby, the exogenous integrase can mediate the integration of the foreign gene into the genome of CHO cells through the attB sequence or attP sequence.

[0015] In some aspects, the present invention provides a system for site-specific integration of antibody gene sequences in the genome of CHO cells, which can integrate the antibody gene sequences into the genome of CHO cells to stably express antibodies in these CHO cells, and it includes:

[0016] a. A vector system comprising a promoter, an antibody heavy chain sequence and a light chain sequence, and attB sequences or attP sequences on both sides thereof; wherein, the antibody heavy chain sequence and the light chain sequence are independently integrated into this vector system and are respectively controlled by independent promoters;

[0017] b. Nucleic acids encoding exogenous integrases Bxb1 or phiC31.

[0018] In some aspects, the present invention provides a kit for site-specific integration of exogenous genes into the genome of CHO cells, which comprises:

[0019] a. A donor vector comprising an exogenous gene region and attB sequences and attP sequences located on both sides of said region;

[0020] b. A vector or nucleic acid encoding phiC31 or Bxb1 integrase. Brief Description of the Drawings

[0021] With reference to the following description, the appended claims and the drawings, some aspects and features of the embodiments described herein will be apparent, wherein:

[0022] Figure 1A Shows a schematic diagram of the principle of site-specific integration according to some embodiments of the present invention. After the integrase induces the integration reaction between the attB and attP sequences on both sides of the mCherry fluorescent protein in the Donor plasmid, the integration efficiency of the four integrases is characterized by the green fluorescent protein (eGFP);

[0023] Figure 1B Shows a schematic diagram of the plasmid map of the integrase vector pCHO-Helper according to some embodiments of the present invention;

[0024] Figure 1C Shows a schematic diagram of the plasmid map of the vector pCHO-Donor expressing the exogenous gene fluorescent protein (mCherry+eGFP) according to some embodiments of the present invention;

[0025] Figure 1D Shows the amount of green fluorescent protein characterizing the expression of eGFP content after the integration reaction of the four integrases according to some embodiments of the present invention;

[0026] Figure 2A Shows a schematic diagram of the plasmid map of the negative control pHY012 empty plasmid according to some embodiments of the present invention;

[0027] Figure 2B Shows a schematic diagram of the plasmid map of the positive control pHY007-green fluorescent protein plasmid according to some embodiments of the present invention;

[0028] Figure 2CShows the cell viability of two control groups and two integrases Bxb1 and phiC31 after expression in

[0029] CHO cells;

[0030] Figure 2D Shows the cell viability density of two control groups and two integrases Bxb1 and phiC31 after expression in CHO cells;

[0031] Figure 3A Shows a schematic diagram of the plasmid map of the linearized (pCGS3-SalI) random integration process of the existing platform according to some embodiments of the present invention;

[0032] Figure 3B Shows a schematic diagram of the plasmid map of supercoiled pCGS3-TrastuzumabDonor according to some embodiments of the present invention;

[0033] Figure 3C Shows a schematic diagram of the plasmid map of supercoiled p375-phiC31Helper according to some embodiments of the present invention;

[0034] Figure 3D Shows the expression of exogenous protein (trastuzumab) in CHO cells of the control group of the linearized (pCGS3-SalI) random integration process and the experimental group of supercoiled pCGS3-TrastuzumabDonor + p375-phiC31Helper according to some embodiments of the present invention;

[0035] Figure 4A Shows the total cell expression of mCherry fluorescent protein in each mutant group of attB and attP of the phiC31 integrase system measured by flow cytometry according to some embodiments of the present invention;

[0036] Figure 4B Shows the positive cell expression of mCherry fluorescent protein in each mutant group of attB and attP of the phiC31 integrase system measured by flow cytometry according to some embodiments of the present invention;

[0037] Figure 4C Shows the total cell expression of mCherry fluorescent protein in each mutant group of attB and attP of the Bxb1 integrase system measured by flow cytometry according to some embodiments of the present invention;

[0038] Figure 4DShows the positive cell expression of mCherry fluorescent protein in each mutant group of attB and attP in the Bxb1 integrase system determined by flow cytometry according to some embodiments of the present invention;

[0039] Figure 5 Shows the protein expression of two monoclonal antibodies, Trastuzumab and Pembrolizumab, under random integration and site-directed integration conditions according to some embodiments of the present invention;

[0040] Figure 6 Shows the protein expression of Trastuzumab under the mediation of Bxb1 mRNA integrase according to some embodiments of the present invention. Detailed implementation manners

[0041] The present invention will be further described in detail below through examples, and should not be construed as a limitation of the present invention.

[0042] I. Definitions

[0043] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to polymers of deoxyribonucleotides or ribonucleotides in linear or circular conformation, and in single-stranded or double-stranded form, which can be DNA or RNA (including mRNA). Generally, analogs of specific nucleotides have the same base-pairing specificity, for example, an analog of A will base-pair with the T base.

[0044] The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to polymers of amino acid residues. This term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

[0045] "Binding" refers to sequence-specific, non-covalent interactions between macromolecules (such as between proteins and nucleic acids). The components of the binding interaction are sequence-specific as a whole.

[0046] "Recombination" refers to the process of genetic information exchange between two polynucleotides, including but not limited to donor capture through non-homologous end joining and homologous recombination. "Homologous recombination" refers to a specialized form of exchange that occurs, for example, during the repair of double-strand breaks in cells via homology-directed repair mechanisms. Homologous recombination usually causes changes in the sequence of the target molecule such that part and / or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0047] The term "sequence" or "nucleic acid sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA (including mRNA), can be linear, circular or branched, and can be single-stranded or double-stranded. The term "donor" or "donor sequence" is a nucleotide sequence inserted into the genome.

[0048] "Chromatin" is the nuclear protein structure that includes the cell genome. Cellular chromatin includes nucleic acids, mainly DNA, and proteins, including histone and non-histone chromosomal proteins. Most eukaryotic cell chromatin exists in nucleosomes, where the nucleosome core includes about 150 base pairs of DNA associated with an octamer.

[0049] "Chromosome" is a chromatin complex that includes all or part of the cell genome. Usually, the genome of a cell is characterized by its karyotype, which is the collection of all chromosomes that make up the cell genome. The genome of a cell can include one or more chromosomes.

[0050] "Target site" or "target sequence" is a nucleic acid sequence that defines the part of a nucleic acid that will bind to a binding molecule under conditions that promote binding.

[0051] "Exogenous" refers to a substance that is not normally present in a cell but can be introduced into the cell by one or more genetic, biochemical or other methods, and "normally present in the cell" is determined in terms of a specific developmental stage and environmental conditions of the cell. For example, exogenous proteins or nucleic acids. For example, exogenous nucleic acids can include infectious virus genomes, plasmids or episomes introduced into cells, or chromosomes that are not normally present in cells. For the purposes disclosed in the present invention, the exogenous nucleic acid can be a plasmid or nucleic acid (such as mRNA, etc.). Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral lipids and cationic lipids), electroporation, direct injection, cell fusion, calcium phosphate co-precipitation, particle bombardment-mediated transfer and virus vector-mediated transfer.

[0052] As used herein, "random integration" generally refers to the integration of an exogenous gene into the CHO genome by a random integration method. Usually, after transfection with an expression plasmid, a cell line with exogenous gene expression or high expression is obtained by pressure screening, but there is uncertainty in the integration site and instability in expression.

[0053] For the purposes of the present disclosure, "gene" includes the DNA region encoding the gene product and all DNA regions that regulate the production of the gene product; thus, a gene includes, but is not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

[0054] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. The gene product can be the direct transcriptional product of the gene (e.g., mRNA, tRNA, rRNA, antisense RAN, ribonuclease, structural RNA, or any other type of RNA) or a protein produced by translating the mRNA.

[0055] "Regulation" of gene expression refers to a change in gene activity. Regulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cutting, altering, inactivating, random mutagenesis) can be used to regulate expression.

[0056] A "vector" is capable of transferring a gene sequence into a target cell. Generally, "vector construct", "expression vector", and "gene transfer vector" mean any nucleic acid construct capable of directing the expression of a gene of interest and transferring the gene sequence into a target cell.

[0057] A "reporter gene" or "reporter sequence" refers to any sequence that produces a protein product that is easily measurable in a conventional assay. For example, suitable reporter genes include, but are not limited to, protein sequences encoding for mediating antibiotic resistance, sequences encoding for colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced fluorescent protein, red fluorescent protein, luciferase), and proteins mediating enhanced cell growth and / or gene amplification.

[0058] "Pseudo gene", also known as a pseudogene, refers to a non-functional copy of a gene that appears in the genome of a specific population, but may contain a segment similar to the known gene sequence in other organisms. For example, there are approximately 10 2 ~10 3 pseudo att sites in mammalian cells. In the embodiments involved in the present invention, a donor plasmid carrying an attB or attP sequence and phiC31 or Bxb1 integrase are introduced into an unmodified cell (CHO cell), and the attB or attP sequence on the donor plasmid can integrate with the pseudo attP or pseudo attB sequence site in the genome of the CHO cell.

[0059] The term "wild type" represents the typical form of a biological organism, strain, or gene, or the characteristics that distinguish it from mutant or variant forms when it exists in nature.

[0060] A "variant" should be understood to represent a form that has been derived from a pattern existing in nature, which can be either naturally occurring or artificially directionally selected.

[0061] As used herein, "trastuzumab" is a monoclonal antibody targeting the HER2 antigen, an antibody comprising a light chain having the nucleotide sequence shown in SEQ ID NO:71 and a heavy chain having the nucleotide sequence shown in SEQ ID NO:72 and the CDRs contained therein; it is understood that the term "trastuzumab" as used herein encompasses "rhuMb4D5" (e.g., the antibody disclosed in U.S. Patent No. 5,821,337) and biosimilars of trastuzumab drugs.

[0062] As used herein, "pembrolizumab", with the trade name Keytruda is a recombinant human IgG4 immunoglobulin that specifically targets the programmed cell death protein-1 (PD-1) receptor. It is an antibody comprising a light chain having the nucleotide sequence shown in SEQ ID NO:73 and a heavy chain having the nucleotide sequence shown in SEQ ID NO:74 and the CDRs contained therein. It is understood that the term "pembrolizumab" as used herein encompasses biosimilars of pembrolizumab.

[0063] The term "antibody" refers to a protein composed of one or more polypeptides encoded substantially by immunoglobulin genes. Currently, the recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes and a large number of immunoglobulin variable region genes. The "antibody" of the present disclosure is used in the broadest sense herein and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, and antibody fragments, and also encompasses antibody conjugates and fusion antibodies.

[0064] II. Overview

[0065] The present invention provides a method for site-specific integration of a foreign gene into the CHO cell genome, which is based on introducing an exogenous integrase and a foreign gene into CHO cells, and the foreign gene is integrated into the CHO cell genome under the action of the exogenous integrase; compared with the existing integration methods, this method has high integration efficiency, high cell consistency, stable integration of the foreign gene, and significantly higher expression level of the foreign gene than that of the random integration control.

[0066] The method of the present invention involves introducing an exogenous integrase-encoding nucleic acid and an exogenous nucleic acid sequence into CHO cells, wherein the exogenous nucleic acid sequence contains a foreign gene and attB sequences or attP sequences located on both sides of the foreign gene, whereby the foreign gene is integrated into the CHO cell genome under the action of the exogenous integrase.

[0067] On the other hand, the present invention also provides a vector system for site-specific integration of exogenous genes into the CHO cell genome, which includes one or more vectors capable of introducing exogenous integrase and exogenous genes into CHO cells.

[0068] III. Integrase and attB, attP sequences

[0069] Integrase can catalyze the cleavage and rejoining of nucleic acid strands at specific sites (or integration sites, attachment sites). According to the nucleophilic active site amino acid residues that attack specific DNA phosphodiesters to cleave the strand, it is divided into serine recombinases and tyrosine recombinases; integrase recognizes sites composed of two inverted repeat binding elements, and these bindings are located on both sides of the spacer sequence (such as exogenous genes) where DNA cleavage and rejoining occur. In the specific embodiments of the present invention, PhiBT1 (Reference 1), PhiRV1 (Reference 2), phiC31 (Reference 3), and Bxb1 (Reference 4) integrases are selected. In the preferred embodiments of the present invention, phiC31 and Bxb1 integrases are selected; and phiC31 and Bxb1 integrases perform DNA cleavage and rejoining through attB and attP recognition sites; generally, the length of the attB and attP recognition sites (core regions) is usually about 30 to 50 nucleotides, and the att sequence specificity of the integrase is determined by two core bases. For example, in the specific embodiments of the present invention, for the wild type, the attB sequence of Bxb1 integrase is shown in SEQ ID NO:1, the attP sequence is shown in SEQ ID NO:18, the attB sequence of phiC31 integrase is shown in SEQ ID NO:35, and the attP sequence is shown in SEQ ID NO:52.

[0070] In some embodiments of the present invention, the integrase is Bxb1, which is a serine recombinase encoded by Mycobacterium phage Bxb1. This serine recombinase can be used to introduce genes of any human, mouse, or other species or synthetic constructs into the mammalian cell genome. Specifically, Bxb1 integrase functions to perform DNA strand exchange between the unique attachment sites of the phage ("attP") and its bacterial host ("attB") during the lytic phase, and the length of each attachment site is about 50 nucleotide base pairs (bp). Although the Bxb1 integrase attachment DNA sites are relatively small, the integration reaction is highly selective for these sites and has strong directionality (see Singh A et al.

[0071] Plos Genetics 2013; 9(5):e1003490), and generally the att sequence specificity of the integrase is determined by two core bases.

[0072] In some embodiments of the present invention, the integrase is phiC31, which is a serine recombinase encoded by a Streptomyces phage. Similar to the above-mentioned Bxb1 enzyme, it can perform DNA strand exchange between the unique attachment sites of the phage ("attP") and the host ("attB"). The core region of the region recognizing the phiC31 site is approximately 35 bp.

[0073] It should be understood that the integrase attachment sites Bxb1 and phiC31 introduced into the host cell genome can be attP sites, mutated or modified attP sites, attB sites, mutated or modified attB sites. The corresponding donor polynucleotide to be inserted into the integrase attachment site should include one or more attachment sites corresponding to this attachment site. Thus, for example, if the integrase attachment site in the genome is an attP site, the corresponding polynucleotide to be inserted into the genome includes an attB site; and if the integrase attachment site in the genome is an attB site, the corresponding polynucleotide to be inserted into the genome includes an attP site. In some specific embodiments of the present invention, the corresponding donor polynucleotide is a plasmid containing an exogenous gene, with attP or attB sites on both sides of the exogenous gene, and the corresponding attachment sites in the CHO cell genome are pesudoattB or pesudoattP sites.

[0074] IV. Method for site-directed integration of exogenous genes

[0075] The present invention specifically provides a method for site-directed integration of exogenous genes into the CHO cell genome. For example, the exogenous genes site-directed integrated into the CHO cell genome encode therapeutic proteins, recombinant proteins or industrial proteins, such as antibodies, etc.

[0076] The method for site-directed integration of exogenous genes into the CHO cell genome provided by the present invention includes introducing an exogenous integrase-encoding nucleic acid and an exogenous nucleic acid sequence into the CHO cell, wherein the exogenous nucleic acid sequence contains an exogenous gene and attB sequences or attP sequences located on both sides of the exogenous gene. Thus, the exogenous gene is integrated into the CHO cell genome under the action of the exogenous integrase. Preferably, the expression level of the thus-integrated exogenous gene is significantly higher than that of the random integration control.

[0077] In some embodiments of the present invention, the method for site-directed integration of exogenous genes into the CHO cell genome includes introducing an exogenous, engineered vector system into the CHO cell. The vector system includes one or more vectors, and one or more vectors contain:

[0078] a. an exogenous gene;

[0079] b. attB sequences or aatP sequences located on both sides of the exogenous gene;

[0080] and / or c. an exogenous integrase-encoding nucleic acid capable of expressing an exogenous integrase in CHO cells;

[0081] wherein a, b, and c may be located in the same or different vectors.

[0082] In some embodiments, the vector system comprises two vectors, one of which contains an exogenous gene and an attB or attP sequence and serves as a donor plasmid (e.g., pCHO-Donor plasmid), and the other contains an integrase-encoding nucleic acid (e.g., phiC31, Bxb1 integrase) and serves as a helper plasmid (e.g., pCHO-Helper plasmid). In some embodiments, the vector system comprises only one vector that simultaneously contains an exogenous gene and an attB or attP sequence and an integrase-encoding nucleic acid; in some specific embodiments of the present invention, the vector system comprises one vector that contains an exogenous gene and an attB or attP sequence, and the mRNA encoding the integrase is introduced into CHO cells.

[0083] Integrase relies on the attB / attP attachment sites when integrating exogenous genes. Therefore, in some embodiments of the present invention, the attB sequence is located on both sides of the exogenous gene, and the exogenous gene is integrated into the corresponding pseudo attP site in the CHO cell genome under the action of the exogenous integrase. In some embodiments of the present invention, the attP sequence is located on both sides of the exogenous gene, and the exogenous gene is integrated into the corresponding pseudo attB site in the CHO cell genome under the action of the exogenous integrase.

[0084] The examples described in the present invention studied the integration efficiency and cell viability of integrases known in the art. In some embodiments, the integrase is phiC31 integrase or Bxb1 integrase. In specific embodiments, the integrase is phiC31 integrase. In other specific embodiments, the integrase is Bxb1 integrase.

[0085] Specific embodiments of the present invention designed mutations in two core bases of the integrase att sequence and studied the expression efficiency of the exogenous genes integrated by the integrase-mediated integration in CHO cells (such as, but not limited to, CHOZN cells), and the mutation design is shown in Table A.

[0086] Table A. att sequence mutation design

[0087]

[0088]

[0089]

[0090]

[0091] Among them, in Table A, the two core bases with att mutations are in the middle of the underlines, and the front and back sequences are the same among different mutant sequence groups.

[0092] In the study on att sequence mutations involved in the present invention, according to the expression efficiency of the foreign gene (GFP), the attB sequence and the attP sequence for integration into the CHO cell genome are screened. In a preferred embodiment of the present invention, the attB sequence of Bxb1 integrase is as shown in SEQ ID NO:11, or the attB sequence of phiC31 integrase is as shown in SEQ ID NO:40.

[0093] V. System for site-specific integration of foreign genes

[0094] The present invention also provides a system for site-specific integration of a foreign gene into the CHO cell genome, which mainly introduces a foreign gene and an exogenous integrase into the CHO cell so that the foreign gene can be integrated into the CHO cell genome under the action of the integrase. Preferably, the expression level of the foreign gene integrated thereby is significantly higher than that of the random integration control.

[0095] In the system for site-specific integration of a foreign gene into the CHO cell genome involved in some embodiments of the present invention, it includes one or more vectors, wherein the vector contains: a. a foreign gene, b. an attB sequence or an attP sequence, located on both sides of the foreign gene; c. a nucleic acid encoding an exogenous integrase. In some embodiments of the present invention, the foreign gene and the exogenous integrase are contained in the same vector, for example, in different coding frames. In other some embodiments of the present invention, the foreign gene and the exogenous integrase are constructed in different vectors. For example, in an exemplary embodiment of the present invention, the foreign gene is constructed in the Donor plasmid, and the exogenous integrase phiC31 or Bxb1 is constructed in the Helper plasmid. Among them, the Donor plasmid and the Helper plasmid can be recombinantly constructed from the existing plasmid pCHO or the commercialized pCGS3-Donor and p375-Helper plasmids.

[0096] In some embodiments, the vector system comprises two vectors, one of which contains an exogenous gene and an attB or attP sequence, serving as a donor plasmid (e.g., pCGS3-Donor plasmid), and the other contains an integrase-encoding nucleic acid (e.g., phiC31, Bxb1 integrase), serving as a helper plasmid (e.g., p375-Helper plasmid). In some embodiments, the vector system comprises only one vector, which simultaneously contains an exogenous gene and an attB or attP sequence as well as an integrase-encoding nucleic acid; in some specific embodiments of the present invention, the vector system comprises one vector, which contains an exogenous gene and an attB or attP sequence, and the mRNA encoding the integrase is introduced into CHO cells.

[0097] In some other embodiments of the present invention, the exogenous gene is constructed in a vector, and the exogenous integrase phiC31 or Bxb1 is introduced into CHO cells as DNA or mRNA. For example, in an exemplary embodiment of the present invention, the exogenous gene is constructed in the Donor plasmid, and the exogenous integrase phiC31 or Bxb1 is constructed in the Helper, and the Helper is constructed on the vector DNA or directly synthesized as mRNA. The DNA sequence encoding Bxb1 is shown in SEQ ID NO:69, and its mRNA sequence is only replacing T with U. The mRNA sequence encoding phiC31 is shown in SEQ ID NO:70, and its mRNA sequence is only replacing T with U.

[0098] Table B. Integrase Bxb1 and phiC31 - DNA sequences

[0099]

[0100]

[0101]

[0102] VI. System for site-specific integration of antibody gene sequences in the CHO cell genome

[0103] Another aspect of the present invention provides a system for site-specific integration of antibody gene sequences in the CHO cell genome, so that the antibody gene sequences can be stably integrated into the CHO cell genome and enable efficient and stable expression of the antibody genes. Preferably, the expression level of the exogenous gene integrated thereby is significantly higher than that of the random integration control. The system for site-specific integration of antibody gene sequences in the CHO cell genome includes:

[0104] a. A vector system comprising a promoter, an antibody heavy chain sequence and a light chain sequence, and attB sequences or attP sequences on both sides thereof; wherein, the antibody heavy chain sequence and the light chain sequence are independently integrated into this vector system and are respectively controlled by independent promoters;

[0105] b. A nucleic acid encoding exogenous integrase Bxb1 or phiC31.

[0106] In a specific embodiment of the present invention, the vector system may further comprise other elements to enable screening and stability when it is transferred into CHO cells, such as antibiotic resistance genes, endonuclease sites, replicons, etc. The above-mentioned vector system sequentially comprises an attB or attP sequence, a promoter, an antibody heavy chain sequence, a promoter, an antibody light chain sequence and the corresponding attB or attP sequence. Among them, the promoter is a promoter commonly used for expressing exogenous proteins in CHO cells in the art, such as the hCMV promoter specifically used in the specific embodiment of the present invention.

[0107] In a specific embodiment of the present invention, the attB sequence of integrase Bxb1 is shown as any one of SEQ ID NO: 2-17, or the attP sequence of integrase Bxb1 is shown as any one of SEQ ID NO: 19-34, or the attB sequence of integrase phiC31 is shown as any one of SEQ ID NO: 36-51, or the attP sequence of integrase phiC31 is shown as any one of SEQ ID NO: 53-68; that is, in a specific embodiment, exogenous integrase Bxb1 is used, and the corresponding attB sequence or attP sequence of integrase Bxb1 is used; in other specific embodiments, exogenous integrase phiC31 is used, and the corresponding attB sequence or attP sequence of integrase phiC31 is used. In a preferred embodiment, the attB sequence is shown as any one of SEQ ID NO: 9-11, or the attB sequence is shown as any one of SEQ ID NO: 40-41.

[0108] In the specific embodiments of the present invention, the nucleic acid encoding the exogenous integrase Bxb1 or phiC31 can be integrated in the vector system a or independent of the vector system a. For example, the integrase Bxb1 or phiC31 is in an independent exogenous vector to be able to be expressed in CHO. In other embodiments of the present invention, the integrase Bxb1 or phiC31 is introduced into CHO cells in the form of mRNA to be able to be expressed in CHO cells. Specifically, in a specific embodiment of the present invention, the mRNA sequence encoding the integrase Bxb1 is as shown in SEQ ID NO:69 (where T is changed to U); in another specific embodiment, the mRNA sequence encoding the integrase phiC31 is as shown in SEQ ID NO:70 (where T is changed to U).

[0109] The following examples specifically illustrate the subject matter of the present invention, but should not be construed as limiting in any way.

[0110] Example 1. Study on the integrative activity of integrase in cells

[0111] This example studies the integration efficiency of four integrases, phiBT1, phiRV1, phiC31, and Bxb1, for foreign genes in CHO cells, specifically characterized by fluorescent proteins (mCherry + eGFP). Referring to Figure 1A ~C, the pCHO-Helper plasmid is used to express the four integrases, phiBT1, phiRV1, phiC31, and Bxb1, and the pCHO-Donor plasmid carries the mCherry fluorescent protein and the eGFP green fluorescence. After these two plasmids are transfected into CHO cells (CHOZN cells), the expressed integrase will induce the integration reaction of the attB and attP sequences on both sides of the mCherry fluorescent protein in the Donor plasmid ( Figure 1A ), resulting in the deletion of the mCherry sequence in the Donor plasmid. Before this integration reaction occurs, only the mCherry red fluorescent protein is expressed in the cells, and the eGFP green fluorescent protein is not expressed; after the integration reaction occurs, only the eGFP green fluorescent protein is expressed in the cells, and the mCherry red fluorescent protein is not expressed; therefore, according to the signal ratio of the mCherry red fluorescent protein and the eGFP green fluorescent protein, the occurrence efficiency of the integration reaction is determined, and the two integrases with the highest integration reaction (phiC31 and Bxb1) are selected.

[0112] Among them, the design of each pair of Donor and Helper plasmids is as follows:

[0113] pCHO-Donor plasmid: hCMV promoter - attB - mCherry - attP - eGFP, where the attB and attP sequences are wild-type (the sequences are referred to Table A),

[0114] pCHO-Helper plasmid: hCMV promoter - 3xFlag - SV40 NLS - Integrase - Nucleoplasmin NLS;

[0115] The plasmid components adopt common sequences in this technical field or are from the Genebank database.

[0116] The construction process of pCHO-Donor plasmid is as follows: The hCMV promoter, attB-mCherry-attP, and eGFP sequences are successively constructed between the EcoRI / HindIII digestion sites of pMD19-T vector in multiple steps.

[0117] The construction process of pCHO-Helper plasmid is as follows: The hCMV promoter, 3xFlag - SV40 NLS (nuclear localization signal) - Integrase - Nucleoplasmin NLS sequences are successively constructed between the EcoRI / HindIII digestion sites of pMD19-T vector in multiple steps.

[0118] The plasmid information used in this example is shown in Table 1.1.

[0119] Table 1.1 Plasmid information for in vivo integrase activity research

[0120]

[0121] The experimental procedure is as follows:

[0122] 1. Cell culture: Resuscitate CHOZN cells in CD CHO Fusion (containing 6 mM glutamine) medium, passage and recover until the viability is above 95% and in the logarithmic growth phase (VCD = 0.5 - 3.0E6 cells / mL), and culture them in suspension in a 37°C shaker (200 rpm, amplitude 25 mm, CO2 content 5%, humidity 80%);

[0123] 2. Electroporation: Use the Neon (Thermo) electroporation system to co-transfect 20 μg of Dornor plasmid and 6 μg of Helper plasmid;

[0124] 3. Recovery: The electroporated cells are statically cultured and recovered in a 6-well plate for 3 days (CO2 content 5%, humidity 80%), and the integration efficiency is measured using a flow cytometer;

[0125] 4. Flow cytometry: Measure the fluorescence of different experimental groups using the FITC and PE channels respectively, and calculate the integration efficiency.

[0126] Flow cytometry results showed that among the four integrases, the positive cell ratios of Bxb1 and phiC31 were relatively high, approximately 73%, and that of phiRV1 was the lowest, 60.07% (Table 1.2). In the analysis of the fluorescence intensity of positive cells, almost no green fluorescence expression was detected in the negative control, and the average fluorescence intensity of the positive control was as high as 1.8×10 6 , and the average fluorescence intensity of the four integrases was ranked as phiC31 > Bxb1 > phiRV1 > phiBT1. Among them, the average fluorescence intensity of phiC31 was approximately 5 times that of phiBT1, and Bxb1 was approximately 3 times that of phiBT1( Figure 1D ).

[0127] Table 1.2 Comparison of positive cell rates of four integrases

[0128]

[0129] Example 2. Cytotoxicity study of phiC31 and Bxb1 integrases on the growth of CHO cells

[0130] In this example, the cytotoxicity of exogenous integrases phiC31 and Bxb1 and plasmids on the growth of CHO cells was studied. The pHY012 empty plasmid was used as the negative control, and the pHY007-green fluorescent protein plasmid was used as the positive control. The plasmid information used in the study is shown in Table 2.1.

[0131] Table 2.1 Plasmid information for integrase cytotoxicity study

[0132]

[0133] The experimental procedure was as follows:

[0134] The negative control pHY012 was an empty plasmid that did not express any protein, and its plasmid information was as Figure 2A shown; the positive control pHY007 expressed green fluorescent protein to exclude the influence of exogenous expressed protein on cell growth, and its plasmid information was as Figure 2B shown. pCHO34 and pCHO35 expressed integrases Bxb1 and phiC31, respectively.

[0135] In CHO cells, 20 μg of Helper plasmids (pCHO34 and pCHO35) expressing phiC31 or Bxb1 integrase and control plasmids were electroporated. The Bulk pool was cultured in a 6-well plate - T25 - T75 - statically until the cell viability recovered to more than 80% (CO2 content 5%, humidity 80%), and then transferred to a 50 mL shake flask for suspension culture until the viability recovered to more than 90%.

[0136] There was no significant difference in the growth curves between CHO cells transfected with phiC31 and Bxb1 and CHO cells with control plasmids. Therefore, these two integrases are not cytotoxic to the growth of CHO cells( Figure 2C , Figure 2D ).

[0137] Example 3. Study on the expression efficiency of integrase supercoiled plasmids and platform linearized plasmids for target genes

[0138] This example studied the expression of exogenous protein (trastuzumab) of the existing linearized (pCGS3-SalI) random integration process of the Henlius platform and the supercoiled pCGS3-Donor vector + p375-phiC31 plasmid in CHO cells.

[0139] The specific experimental design is shown in Table 3.1

[0140] Table 3.1 Information of experimental groups

[0141]

[0142] Note:

[0143] Among them, the group of linearized trastuzumab plasmid (pCGS3-Trastuzumab-SalI) is the current random integration process, that is, the control group, and the experimental group is: supercoiled trastuzumab plasmid pCGS3-Donor + p375-phiC31 Helper plasmid. The nucleotide sequence of trastuzumab is shown in Table 5.2. Referring to Figure 3A ~D, in this study, the expression level of exogenous protein (trastuzumab) of the site-specific integration experimental group of trastuzumab plasmid pCGS3-Donor + p375-phiC31 Helper was comparable to that of the group of linearized trastuzumab plasmid (pCGS3-Trastuzumab-SalI). Therefore, the integrase CHO cell transfection process was successfully explored: 20 μg of pCGS3-Donor and 6 μg of p375-Helper plasmid were co-transfected into CHO cells.

[0144] The experimental procedure is as follows:

[0145] 1. Cell culture: Resuscitate CHOZN cells in CD CHO Fusion (containing 6 mM Glutamine) medium, passage and recover to a viability of more than 95%, and be in the logarithmic growth phase (VCD = 0.5 - 3.0E6 cells / mL), and suspend culture in a 37°C shaker (200 rpm, amplitude 25 mm, CO2 content 5%, humidity 80%);

[0146] 2. Electroporation: Using the Neon (Thermo) electroporation system, co-transfect 20 μg of the Dornor plasmid and 6 μg of the Helper plasmid;

[0147] 3. Recovery: The electroporated cells are statically cultured in 6-well plates - T25 - T75 (37 °C, 5% CO2 content, 80% humidity) until the viability is over 80%, and then inoculated into a 50 mL shake flask for suspension culture until the viability is restored to over 90% (200 rpm, amplitude 25 mm, 37 °C, 5% CO2 content, 80% humidity);

[0148] 4. Fed-batch culture: Using the HM005 + 6 g / L PFACF basal medium, in combination with the 0.6xIs Feed 4 feeding medium for Fed-batch culture (200 rpm, amplitude 25 mm, 37 °C, 5% CO2 content, 80% humidity). Starting from the third day, add 5% of the current volume of the 0.6xIs Feed 4 feeding medium every day. When VCD > 10E6 cells / mL, lower the temperature to 37 °C and culture until day 15 for harvest.

[0149] 5. Antibody yield determination: Determine the standard curve, and after centrifugation, take the samples in the supernatant of the culture medium and detect the target protein yield by HPLC.

[0150] Example 4. Study on the mutation of integrase att sequences

[0151] This example studies the mutations of the attB / P sequences corresponding to the integrases phiC31 and Bxb1, and obtains the preferred attB / attP sequence combination through the integration efficiency and expression efficiency of the foreign gene (GFP protein) in the CHO cell genome caused by the mutations.

[0152] The specificity of the integrase att sequence is determined by its core two bases. For example, if the core two bases of attB and attP are the same, the integration reaction can occur; otherwise, it cannot. For a specific integrase system, the attB sequence can be designed on the vector and integrated into the pseudoattP site of the CHO cell genome under the mediation of the integrase, or the attP sequence can be designed on the vector and integrated into the pseudo attB site of the CHO cell genome under the mediation of the integrase. Due to the core two bases, each base has four possibilities: A / T / C / G, so there are 16 combinations for two bases. At the same time, according to whether attB / P is designed on the vector, there are 32 combinations.

[0153] Since DNA is a double-stranded helix with A / T and C / G complementary base pairing, for example, when designing the attB(AA) and attB(TT) sequences on the vector, their integration sites in the CHO cell genome are the same, only the integration directions are different. Therefore, similar experimental groups can be combined to produce 20 combinations. The specific experimental groups and the wild-type and mutant sequences of attB and attP of the integrases phiC31 and Bxb1 are shown in Table A.

[0154] The experimental procedure is as follows:

[0155] Cell culture: Resuscitate CHOZN cells in CD CHO Fusion (containing 6 mM glutamine) medium, passage and recover until the viability is above 95% and in the logarithmic growth phase (VCD = 0.5 - 3.0E6 cells / mL), and suspend culture in a 37°C shaker (200 rpm, amplitude 25 mm, CO2 content 5%, humidity 80%);

[0156] Electroporation: Using the Neon (Thermo) electroporation system, co-transfect 20 μg of pCHO-Donor plasmid and 6 μg of pCHO-Helper plasmid (for plasmid information, refer to Example 1, the mCherry gene is integrated between the attB or attP sequences);

[0157] Recovery: The electroporated cells are statically cultured and recovered in a 6-well plate for 3 days (CO2 content 5%, humidity 80%), and the integration efficiency is measured using a flow cytometer;

[0158] Flow cytometry: Use a flow cytometer to measure the expression of mCherry fluorescence and calculate the integration efficiency. The results show that for both the phiC31 and Bxb1 integrase systems, the integration scheme with the mutated attB sequence designed on the vector has the highest expression efficiency of foreign genes. Refer to Figure 4A - Figure 4D As shown, integrase-mediated foreign gene integration ( Figure 4B , 4D ) has a much higher positive rate than random integration ( Figure 4A , Figure 4C ); and, referring to Figure 4B and 4D As shown, placing the attB sequence on the Donor plasmid results in better expression of foreign genes, and the optimal attB mutant group has a 40% increase in the fluorescence intensity of positive cells compared to random integration. For example, refer to Figure 4BAs shown, the integration of foreign genes mediated by phiC31-attB-AT mutation and phiC31-attB-TA mutation enhanced the expression of foreign genes by 40% compared with random integration; among them, the integration of foreign genes mediated by Bxb1-attB-GC mutation and Bxb1-attB-TC (or GA) mutation enhanced the expression of foreign genes by 40% compared with random integration. The difference between the integrase group and the control group was analyzed by t-test, and the data were expressed as mean±SD (n = 3), **p<0.01.

[0159] Table 4 Preferred attB sequences

[0160]

[0161] Example 5. Study on the expression level of foreign genes mediated by integrase

[0162] Compared with the current method of random integration of foreign genes in CHO cells, this example studied the integration of the above-mentioned integrase-mediated foreign genes into the CHO cell genome and the expression levels of their Bulk pool foreign genes (Trastuzumab and Pembrolizumab). In this example, SEQ ID NO:40 was used as an exemplary phiC31-attB.

[0163] Among them, 20 μg of linearized Trastuzumab plasmid (Trastuzumab SalI) was used for the current random integration process (control group), and the combination of 20 μg of supercoiled Trastuzumab plasmid pCGS3-Donor + 10 μg of phiC31 p375-Helper plasmid and the combination of 20 μg of supercoiled Trastuzumab plasmid pCGS3-Donor plasmid + 20 μg of phiC31 p375-Helper plasmid were used for the site-specific integration process. In contrast, the combination of 20 μg of supercoiled Trastuzumab plasmid pCGS3-Donor plasmid + 10 μg of phiC31 p375-Helper plasmid increased the yield by 32.14% compared with the random integration group, while the combination of 20 μg of Trastuzumab pCGS3-Donor plasmid + 20 μg of phiC31 p375-Helper plasmid increased the yield by 21.43% compared with the random integration group.

[0164] Among them, 20 μg of linearized Pembrolizumab plasmid (Pembrolizumab SalI) was used for the current random integration process (control group), and the combination of 20 μg of Pembrolizumab Donor plasmid + 10 μg of phiC31 Helper plasmid was used for the site-specific integration process. In contrast, the site-specific integration group increased the yield by 130% compared with the random integration group ( Figure 5 and Table 5.1).

[0165] Table 5.1 Expression levels of trastuzumab or pembrolizumab in each group

[0166]

[0167] The plasmid information for each experimental group is as follows:

[0168] Trastuzumab (SalI): hCMV promoter - light chain - hCMV promoter - heavy chain - SalI site

[0169] Trastuzumab (pCGS3 - Donor): hCMV promoter - light chain - hCMV promoter - heavy chain - attB(at) sequence. Pembrolizumab (SalI): hCMV promoter - light chain - hCMV promoter - heavy chain - SalI site

[0170] Pembrolizumab (pCGS3 - Donor): hCMV promoter - light chain - hCMV promoter - heavy chain - attB(at) sequence

[0171] phiC31 integrase (p375 - Helper): T7 promoter - phiC31 - NLS

[0172] The nucleotide sequences of trastuzumab and pembrolizumab (Pembrolizumab or Keytruda) are shown in Table 5.2:

[0173] Table 5.2 Nucleotide sequences of trastuzumab and pembrolizumab

[0174]

[0175]

[0176] The experimental procedure is as follows:

[0177] 1. Cell culture: Resuscitate CHOZN cells in CD CHO Fusion (containing 6 mM glutamine) medium, passage and recover until the viability is above 95% and in the logarithmic growth phase (VCD = 0.5 - 3.0E6 cells / mL), and culture in suspension in a 37°C shaker (200 rpm, amplitude 25 mm, CO2 content 5%, humidity 80%);

[0178] 2. Electroporation: Use the Neon (Thermo) electroporation system to co - transfect 20 μg of Dornor plasmid and 10 - 20 μg of Helper plasmid;

[0179] 3. Recovery: The electrotransformed cells were statically cultured in 6-well plates - T25 - T75 (37°C, 5% CO2 content, 80% humidity) until the viability was over 80%, and then inoculated into a 50 mL shake flask for suspension culture until the viability recovered to over 90% (200 rpm, amplitude 25 mm, 37°C, 5% CO2 content, 80% humidity);

[0180] 4. Fed-batch culture: Using HM005 + 6 g / L PFACF basal medium, combined with 0.6xIs Feed 4 feeding medium for Fed-batch culture (200 rpm, amplitude 25 mm, 37°C, 5% CO2 content, 80% humidity), starting from day 3, 5% of the current volume of 0.6xIs Feed 4 feeding medium was added every day. When VCD > 10E 6 cells / mL, the temperature was lowered to 37°C and cultured until day 15 for harvest.

[0181] 5. Antibody yield determination: The standard curve was determined, and the samples in the supernatant of the culture medium after centrifugation were used to detect the target protein yield by HPLC method.

[0182] Example 6 Study on the Expression of Exogenous Genes Mediated by mRNA Expression Integrase

[0183] This example studied the Bulkpool yield of the site-specific integration process and random integration process of exogenous genes mediated by integrase, in which Bxb1 integrase was expressed as mRNA, replacing the pCGS3-Helper plasmid in the above examples. This example used SEQ ID NO:40 as the exemplary phiC31-attB.

[0184] 20 μg of linearized trastuzumab plasmid (Trastuzumab SalI) was used for the current random integration process, while 20 μg of supercoiled trastuzumab plasmid pCGS3-Donor DNA + 10 μg of Bxb1 mRNA was used for the site-specific integration process of the present invention. Refer to Figure 6 and Table 6.1, the combination of 20 μg Donor DNA + 10 μg Bxb1 mRNA increased the yield by 39.29% compared with the random integration process ( Figure 6 ).

[0185] Table 6.1 Trastuzumab expression level

[0186]

[0187] The experimental procedure is as follows:

[0188] 1. Cell culture: Resuscitate CHOZN cells in CD CHO Fusion (containing 6 mM Glutamine) medium, passage and recover until the viability is above 95% and in the logarithmic growth phase (VCD = 0.5 - 3.0E6 cells / mL), and suspend culture in a shaker at 37°C (200 rpm, amplitude 25 mm, CO2 content 5%, humidity 80%);

[0189] 2. Electroporation: Use the Neon (Thermo) electroporation system to co-transfect 20 μg of Dornor plasmid and 10 μg of mRNA;

[0190] 3. Recovery: After electroporation, the cells are statically cultured in a 6-well plate - T25 - T75 (37°C, CO2 content 5%, humidity 80%) until the viability is above 80%, and then inoculated into a 50 mL shake tube for suspension culture until the viability is restored to above 90% (200 rpm, amplitude 25 mm, 37°C, CO2 content 5%, humidity 80%);

[0191] 4. Fed-batch culture: Use the HM005 + 6 g / L PFACF basal medium and 0.6xIs Feed 4 feeding medium for Fed-batch culture (200 rpm, amplitude 25 mm, 37°C, CO2 content 5%, humidity 80%). Starting from day 3, add 5% of the current volume of 0.6xIs Feed 4 feeding medium every day. When VCD > 10E 6 cells / mL, cool down to 37°C and culture until harvest on day 15.

[0192] 5. Antibody yield determination: Determine the standard curve, and after centrifugation, take the samples in the supernatant of the culture medium and detect the target protein yield by HPLC method.

[0193] It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0194] Reference 1: Streptomycin phage Catalytic mechanism of integrase-mediated site-specific recombination system and its application in synthetic biology

[0195] Reference 2: Integration and excision of the Mycobacterium tuberculosis prophage-like element, Molecular Microbiology, 2002, 45(6): 1515 - 1526

[0196] Reference 3: Multiple novel promoters from the early region in the Streptomyces temperate phase are activated during lytic development. Molecular Microbiology, 1993, 9(6): 1267 - 1274

[0197] Reference 4: Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Molecular Microbiology, 2003, 50(2): 463 - 473。

Claims

1. A method for site-specific integration of a foreign gene into the genome of CHO cells, which comprises introducing an exogenous integrase-encoding nucleic acid and an exogenous nucleic acid sequence into CHO cells, wherein the exogenous nucleic acid sequence contains a foreign gene and attB sequences or attP sequences located on both sides of the foreign gene, whereby the foreign gene is integrated into the genome of CHO cells under the action of the exogenous integrase, and the expression level of the thus-integrated foreign gene is significantly higher than that of the random integration control.

2. The method according to claim 1, which comprises introducing an exogenous, engineered vector system into CHO cells, the vector system comprising one or more vectors, wherein one or more vectors contain: a. a foreign gene; b. attB sequences or attP sequences located on both sides of the foreign gene; and / or c. an exogenous integrase-encoding nucleic acid, which can express an exogenous integrase in CHO cells; Among them, a, b, and c can be located in the same or different vectors.

3. The method according to claim 1 or 2, wherein the attB sequences are located on both sides of the foreign gene, and the foreign gene is integrated into the genome of CHO cells under the action of the exogenous integrase.

4. The method according to claim 1 or 2, wherein the attP sequences are located on both sides of the foreign gene, and the foreign gene is integrated into the genome of CHO cells under the action of the exogenous integrase.

5. The method according to any one of the preceding claims, wherein the integrase is phage phiC31 integrase or Bxb1 integrase.

6. The method according to any one of the preceding claims, wherein the attB sequence and / or attP sequence is optimized.

7. The method according to any one of the preceding claims, wherein the attB sequence of the integrase Bxb1 is shown as any one of SEQ ID NO: 2-17, or the attP sequence of the integrase Bxb1 is shown as any one of SEQ ID NO: 19-34; or, wherein the attB sequence of the integrase phiC31 is shown as any one of SEQ ID NO: 36-51, or the attP sequence of the integrase phiC31 is shown as any one of SEQ ID NO: 53-68.

8. The method according to any one of the preceding claims, wherein the exogenous integrase is introduced into the cells by RNA, preferably by mRNA.

9. The method according to any one of the preceding claims, wherein the CHO cells are CHOZN cells.

10. The method according to any one of the preceding claims, wherein the foreign gene encodes a therapeutic protein, preferably an antibody, more preferably a monoclonal antibody or a multispecific antibody.

11. A CHO cell prepared by the method according to any one of claims 1-10, which contains a site-specifically integrated foreign gene in its genome.

12. A system for site-specific integration of a foreign gene into the genome of CHO cells, the system comprising one or more vectors, wherein one or more vectors contain: a. a foreign gene; b. attB or attP sequences flanking the exogenous gene; and / or c. exogenous integrase-encoding nucleic acid, which is capable of expressing exogenous integrase in CHO cells; Among them, a, b, and c may be located on the same or different vectors; whereby the exogenous integrase is capable of mediating the integration of the exogenous gene into the CHO cell genome through the attB or attP sequence.

13. The system according to claim 12, which comprises two vector systems, wherein one vector system comprises: a. an exogenous gene; b. attB or attP sequences flanking the exogenous gene; and, the other vector system comprises exogenous integrase-encoding nucleic acid.

14. The system according to claim 12 or 13, which comprises one vector system, which comprises: a. an exogenous gene; b. attB or attP sequences flanking the exogenous gene; and, the exogenous integrase is encoded by exogenous RNA in CHO cells, preferably encoded by exogenous mRNA in CHO cells.

15. The system according to any one of claims 12 - 14, wherein, The exogenous integrase-encoding nucleic acid encodes phiC31 integrase or BxB1 integrase.

16. The system according to claim 14 or 15, wherein the mRNA sequence of the Bxb1 integrase is as shown in SEQ ID NO:69, or the mRNA sequence of the phiC31 integrase is as shown in SEQ ID NO:

70.

17. The system according to any one of claims 12 - 16, wherein, The attB or attP sequence is optimized; preferably, the attB sequence of the Bxb1 integrase is as shown in any one of SEQ ID NOs:2-17, or the attP sequence of the Bxb1 integrase is as shown in any one of SEQ ID NOs:19-34; alternatively, the attB sequence of the phiC31 integrase is as shown in any one of SEQ ID NOs:36-51, or the attP sequence of the phiC31 integrase is as shown in any one of SEQ ID NOs:53-68.

18. The system according to any one of claims 12-17, wherein the attB sequence is as shown in any one of SEQ ID NOs:9-11, or the attB sequence is as shown in any one of SEQ ID NOs:40-41.

19. A system for site-specific integration of an antibody gene sequence into the CHO cell genome, which is capable of integrating the antibody gene sequence into the CHO cell genome for stable expression of the antibody in the CHO cells, and which comprises: a. a vector system, which comprises a promoter, an antibody heavy chain sequence and a light chain sequence, and attB or attP sequences flanking them; wherein, the antibody heavy chain sequence and the light chain sequence are independently integrated into this vector system and are respectively controlled by independent promoters; b. nucleic acid encoding exogenous integrase Bxb1 or phiC31.

20. The system according to claim 19, wherein The attB sequence of the integrase Bxb1 is as shown in any one of SEQ ID NOs:2-17, and the attP sequence of the integrase Bxb1 is as shown in any one of SEQ ID NOs:19-34; Alternatively, the attB sequence of integrase phiC31 is as shown in any one of SEQ ID NOs: 36-51, and the attP sequence of integrase phiC31 is as shown in any one of SEQ ID NOs: 53-68; Preferably, the attB sequence is as shown in any one of SEQ ID NOs: 9-11, or the attB sequence is as shown in any one of SEQ ID NOs: 40-41.

21. The system according to claim 19 or 20, wherein, The nucleic acid encoding exogenous integrase Bxb1 or phiC31 is an exogenous vector or mRNA.

22. The system according to any one of claims 19-21, wherein the mRNA sequence encoding integrase Bxb1 is as shown in SEQ ID NO: 69, or the mRNA sequence encoding integrase phiC31 is as shown in SEQ ID NO:

70.

23. A kit for site-specific integration of an exogenous gene into the genome of CHO cells, comprising: a. A donor vector comprising an exogenous gene region and attB and attP sequences flanking said region; b. A vector or mRNA encoding integrase phiC31 or Bxb1; wherein the attB sequence of integrase Bxb1 is as shown in any one of SEQ ID NOs: 2-17, or the attP sequence of integrase Bxb1 is as shown in any one of SEQ ID NOs: 19-34; Alternatively, the attB sequence of integrase phiC31 is as shown in any one of SEQ ID NOs: 36-51, or the attP sequence of integrase phiC31 is as shown in any one of SEQ ID NOs: 53-68; Preferably, the attB sequence is as shown in any one of SEQ ID NOs: 9-11, or the attB sequence is as shown in any one of SEQ ID NOs: 40-41.

24. The kit according to claim 23, further comprising a nuclease, a ligase, and / or instructions for use.

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