Transgenic animal antibody high-yield expression system based on double-copy site-specific integration and application thereof

Abstract

The invention discloses a transgenic animal antibody high-yield expression system based on double-copy site-specific integration and application of the transgenic animal antibody high-yield expression system. According to the invention, dual-copy anti-staphylococcus aureus enterotoxin B (SEB) monoclonal antibody genes are integrated at safe sites of mouse ROSA26 and H11 in a targeting manner, so that the whole-body high-level stable expression of the antibody in a mouse body is realized, and the yield and neutralizing activity of the antitoxin antibody are remarkably improved. The invention provides a new thought and a new strategy for the development of a low-cost and high-efficiency therapeutic antibody bioreactor. Besides, the constructed SEB antibody gene animal model provides an important tool for research on staphylococcus aureus infection resistance, provides a potential model animal solution for disease prevention and control of animal husbandry, and has good application value and industrial transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a transgenic animal antibody high-yield expression system based on dual-copy site-directed integration and its applications. Background Technology

[0002] Recombinant monoclonal antibodies (mAbs) are widely used in the treatment of cancer, inflammation, and infections. Currently, the mainstream method for producing mAbs is the large-scale culture of mammalian cells (such as hamster ovary cells and CHO cells), but this method is complex, costly (approximately $100 / gram), and has limited production capacity, making it difficult to meet the growing clinical demand. Therefore, there is an urgent need in this field to develop a low-cost alternative production platform technology.

[0003] Transgenic animal bioreactors have shown great potential as an alternative. Their production costs are significantly reduced (only $0.02-0.5 / gram), and the development cycle is shorter, typically 1-2 years. For example, mammary gland bioreactors offer significant advantages; transgenic dairy cows can produce 8,000 liters of milk annually, with each cow producing over 15 kg of mAbs per year, and production capacity can be exponentially expanded through conventional breeding. Mammary glands can perform post-translational modifications on target proteins, ensuring the biological activity and structural stability of antibodies. Studies have confirmed the successful expression of high concentrations of biologically active recombinant humanized antibodies, such as anti-hepatitis A virus (HAV) antibodies, anti-hepatitis B virus (HBV) antibodies, and anti-CD20 antibodies, in the milk of transgenic mice and dairy cows, with affinity and neutralizing activity even superior to antibodies expressed in CHO cells. Furthermore, silkworm silk glands and chicken egg white systems are also considered promising bioreactor platforms: silkworm silk glands offer rapid and low-cost protein synthesis, and can produce humanized mAbs with enhanced ADCC activity. Chicken systems, as bioreactors, have short generation times and high reproductive capacity. They can achieve efficient expression of target genes in egg white through the ovalbumin promoter, and the resulting functional mAbs have similar binding characteristics to commercially available antibodies.

[0004] Despite the immense potential of transgenic bioreactor technology, its application still faces technological bottlenecks, including: unstable expression due to random integration of exogenous genes, gene silencing, low animal utilization, long development cycles (establishing stable transgenic lines requires several years), and stringent regulatory challenges. These bottlenecks limit large-scale production and commercialization. Compared to traditional random transgenic animal bioreactor technology, this patented technology overcomes these bottlenecks through three major innovations, significantly improving efficiency, safety, and economy: First, addressing the defects of random integration (such as insertion mutations, multiple copy variations, and host health risks), this technology utilizes CRISPR / Cas9 gene editing combined with a "safe site" strategy to precisely integrate human mAb genes into mouse ROSA26 or H11 (Hipp11) sites, ensuring continuous and stable gene expression without interfering with host physiological functions, thus improving overall safety. Second, overcoming the limitation of low exogenous antibody concentrations in traditional bioreactors, this technology introduces a multiple copy integration design (2 copies for heterozygotes, 4 copies for homozygotes), significantly increasing expression levels by 10-20 times compared to single copies, significantly improving antibody yield per animal and economic benefits. Third, traditional technologies are limited to expression in specific tissues (such as blood, milk, saliva, or semen), resulting in low animal utilization (for example, mammary gland bioreactors are only usable by females during lactation). This technology, for the first time, employs a systemic expression promoter (CAG), enabling efficient expression of the mAb gene in all sexes, life stages, and multiple tissues of mice, significantly improving animal utilization and production continuity. These innovations not only address the pain points of existing technologies but also provide a reliable foundation for the industrial application of transgenic animal bioreactors, possessing broad clinical and commercial prospects. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems existing in the field, the purpose of this invention is to provide a high-yield expression system for transgenic animal antibodies based on dual-copy site-directed integration and its application.

[0006] This invention inserts two copies of a monoclonal antibody gene (LXY-Ab) against Staphylococcus aureus enterotoxin B (SEB) into the safe sites of ROSA26 and / or H11 (Hipp11) in mice to achieve high systemic expression of this antibody in mice. A preliminary bioreactor for systemic expression of this antibody in mice was prepared. The donor vector construction strategy is as follows: the mammalian high-expression promoter CAG was selected, followed by a signal peptide-light chain-IRES element-signal peptide-heavy chain, and then a copy was repeated as a targeting vector, with approximately 2 kb homologous arms on both sides. PCR and Sanger sequencing also confirmed successful insertion into these two safe sites. ELISA and Biacore analysis showed that the antibodies in serum and CHO expression had comparable affinity. Regarding in vivo protective effects, the antibodies in the serum of the transgenic animals of this invention exhibited superior neutralizing effect against SEB challenge.

[0007] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0008] The first aspect of the present invention provides a transgenic animal antibody high-yield expression system based on dual-copy integration.

[0009] Furthermore, the system includes targeted integration of a target antibody expression cassette into the ROSA26 safe site and / or H11 safe site in mammals, respectively. The target antibody expression cassette comprises a mammalian high-expression promoter CAG, a signal peptide-the target antibody light chain-IRES element-signal peptide-the target antibody heavy chain connected in sequence, and the signal peptide-the target antibody light chain-IRES element-signal peptide-the target antibody heavy chain repeats one copy to form a double-copy structure.

[0010] Furthermore, the mammalian high-expression promoter CAG is further provided with a Kozak sequence downstream, and the target antibody expression cassette is provided with a β-globin polyadenylate signal at its end;

[0011] Optionally, the targeted integration of the ROSA26 safe site is achieved via sgRNA with a sequence as shown in SEQ ID NO:3;

[0012] Optionally, the targeted integration of the H11 safe site is achieved via sgRNA with a sequence as shown in SEQ ID NO:4.

[0013] Furthermore, the target antibody is a monoclonal antibody against Staphylococcus aureus enterotoxin B;

[0014] Optionally, the amino acid sequence of the heavy chain variable region of the monoclonal antibody against Staphylococcus aureus enterotoxin B is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14.

[0015] Optionally, the mammal is a mouse, rat, guinea pig, hamster, pig, cow, sheep, horse, rabbit, or monkey.

[0016] The core innovation of the expression system in this invention lies in the synergistic design of "dual safety sites + dual-copy expression cassettes". Specifically, through gene editing technologies such as CRISPR / Cas9, a set of target antibody expression cassettes is targeted and integrated into two verified "neutral integration sites" (which do not affect the function of other genes in the host genome and have no risk of insertional inactivation) in the mammalian genome. This forms an integration mode of "each site containing a dual-copy expression cassette". The two sets of expression cassettes function independently but synergistically improve antibody yield. By ensuring high antibody production and stability from both the selection of integration sites and the copy number of expression cassettes, unexpected technical effects have been achieved, providing an industrializable technical solution for the low-cost, large-scale preparation of therapeutic antibodies.

[0017] Among them, the ROSA26 safety site is a classic safety site widely used in mammalian genomes. Its first intron region has the characteristics of high transcriptional activity and stable expression of the target gene after integration, which can ensure the continuous expression of the target antibody in multiple tissues throughout the body (such as serum, saliva, mammary glands and other body fluids). The H11 safety site, as another type of highly efficient safety site, has the same "neutral integration" characteristics as the ROSA26 site. Moreover, the independent location of the two in the genome can avoid the risk of gene silencing that may be caused by the integration of multiple copies of a single safety site, further improving the reliability of the expression system.

[0018] In this invention, the Staphylococcus aureus enterotoxin B (SEB) is a typical heat-stable exotoxin produced by pathogenic Staphylococcus aureus (mainly certain toxin-producing strains, such as Staphylococcus aureus FRI 100). It belongs to the superantigen family and possesses both food poisoning induction ability and strong immune activation characteristics. Its molecular structure is stable, exhibiting strong resistance to heat (conventional cooking temperatures are insufficient for complete inactivation, requiring heating at 100°C for several minutes or more) and gastric acid. It is also not easily degraded by digestive enzymes, allowing it to remain stably in contaminated food and enter the human body through the digestive tract. SEB can non-specifically activate a large number of T lymphocytes without the need for a conventional antigen presentation process, triggering an excessive immune response, releasing large amounts of cytokines, and consequently leading to toxic shock syndrome, organ damage, and other severe systemic inflammatory reactions.

[0019] A second aspect of the present invention provides a Donor carrier assembly for constructing the system described in the first aspect of the present invention.

[0020] Furthermore, the Donor vector combination includes a ROSA26 site Donor vector and / or an H11 site Donor vector;

[0021] The ROSA26 site Donor vector comprises, in sequence, a 5′ homologous arm, a CAG promoter, a Kozak sequence, a signal peptide-light chain variable region VK-human light chain constant region hIgK-CL-IRES2 element-signal peptide-heavy chain variable region VH-human heavy chain constant region hlgG1-CH, a signal peptide-light chain variable region VK-human light chain constant region hIgK-CL-IRES2 element-signal peptide-heavy chain variable region VH-human heavy chain constant region hlgG1-CH, a β-globin poly (A) signal, and a 3′ homologous arm;

[0022] The H11 site Donor vector comprises, in sequence, a 5′ homologous arm, a CAG promoter, a Kozak sequence, a signal peptide-light chain variable region VK-human light chain constant region hIgK-CL-IRES2 element-signal peptide-heavy chain variable region VH-human heavy chain constant region hlgG1-CH, a signal peptide-light chain variable region VK-human light chain constant region hIgK-CL-IRES2 element-signal peptide-heavy chain variable region VH-human heavy chain constant region hlgG1-CH, a β-globin poly (A) signal, and a 3′ homologous arm.

[0023] Furthermore, the nucleotide sequence of the ROSA26 site Donor vector is shown in SEQ ID NO:1;

[0024] The nucleotide sequence of the Donor vector at the H11 site is shown in SEQ ID NO:2.

[0025] In this invention, the Donor refers to the donor vector, which is a complete vector molecule designed to achieve targeted integration of the target antibody expression cassette into a specific safe site in the mouse genome. Its core function is to serve as a "homologous targeted repair template" to precisely insert the target antibody expression cassette into the ROSA26 or H11 safe site in the mouse genome under the mediation of the CRISPR / Cas9 system.

[0026] In a specific embodiment of the present invention, Donor is the key vector for achieving the "dual-copy integration and high antibody production" of the present invention: when constructing transgenic mice, Donor, Cas9 protein, and sgRNA targeting the ROSA26 / H11 site are co-injected into mouse zygotes. The Cas9-sgRNA complex first cleaves the genomic target site, and Donor serves as a repair template. Through homologous recombination, the dual-copy expression cassette of the target antibody is inserted into the target site, ultimately achieving high systemic expression of the target antibody in mice (serum concentration as high as 24.71-38.16 mg / mL, more than 10 times higher than single copy).

[0027] A third aspect of the present invention provides a method for constructing the system described in the first aspect of the present invention.

[0028] Furthermore, the method includes the following steps:

[0029] (1) Design and synthesize sgRNA targeting the ROSA26 safe site and / or sgRNA targeting the H11 safe site;

[0030] (2) Construct the ROSA26 site Donor vector and / or H11 site Donor vector as described in the second aspect of the present invention;

[0031] (3) Obtain fertilized eggs from the uterus of the donor female mouse;

[0032] (4) Mix Cas9 protein, sgRNA targeting the ROSA26 safe site and / or sgRNA targeting the H11 safe site, ROSA26 site Donor vector and / or H11 site Donor vector to form an injection mixture, dilute it and draw it into the injection needle, and inject it into the nucleus and cytoplasm of the fertilized egg one by one.

[0033] (5) The injected fertilized eggs were cultured in vitro to the 2-cell stage;

[0034] (6) Select a suitable female mouse and a vasectomized male mouse to obtain a surrogate female mouse, and transplant the fertilized eggs cultured to the 2-cell stage into the uterus of the surrogate female mouse;

[0035] (7) When the surrogate mother mouse gives birth, the genomic DNA of the 1-week-old mice is extracted by cutting off the toes. The positive mice obtained are the high-yield expression system carrying the target antibody.

[0036] Optionally, in step (4), the concentration of Cas9 protein in the injection mixture is 10-50 ng / μL, the concentrations of sgRNA targeting the ROSA26 safe site and the sgRNA targeting the H11 safe site are 2-7 ng / μL and 2-7 ng / μL, respectively, and the concentrations of the ROSA26 site Donor vector and the H11 site Donor vector are 5-15 ng / μL and 5-15 ng / μL, respectively.

[0037] Optionally, in step (4), the concentration of Cas9 protein in the injection mixture is 30 ng / μL, the concentrations of sgRNA targeting the ROSA26 safe site and the sgRNA targeting the H11 safe site are 5 ng / μL and 5 ng / μL, respectively, and the concentrations of the ROSA26 site Donor vector and the H11 site Donor vector are 10 ng / μL and 10 ng / μL, respectively.

[0038] Furthermore, in step (5), the conditions for in vitro culture of fertilized eggs are 37°C, 5% CO2, and the culture medium is M16 medium.

[0039] In this invention, the method is designed with the goal of "constructing a high-yield antibody bioreactor at low cost and high efficiency." Addressing the core requirement of "dual-safety site integration" in the first aspect of the system, it employs CRISPR / Cas9-mediated homology-directed repair (HDR) technology—using sgRNA to guide Cas9 protein to cleave genomic target sites, and then using the Donor vector combination as a repair template to precisely insert the dual-copy antibody expression cassette into the ROSA26 and H11 sites. The entire process primarily uses mice as experimental subjects, but can also be adapted to other mammals such as rats, pigs, and rabbits (species-specific parameters, such as sgRNA sequence and homologous arm sequences, can be routinely adjusted). Finally, transgenic animals carrying the target expression system are obtained through embryo transfer and positive selection.

[0040] A fourth aspect of the present invention provides a transgenic animal bioreactor.

[0041] Furthermore, the transgenic animal bioreactor is a transgenic animal bioreactor prepared using the method described in the third aspect of this invention.

[0042] The fifth aspect of the present invention provides for application in any of the following aspects:

[0043] (1) The application of the system described in the first aspect of the present invention or the ROSA26 site Donor vector and / or H11 site Donor vector described in the second aspect of the present invention in the preparation of anti-target antibody-related disease therapeutic drugs;

[0044] (2) The application of the system described in the first aspect of the present invention or the ROSA26 site Donor vector and / or H11 site Donor vector described in the second aspect of the present invention in constructing a transgenic animal antibody high-yield expression system based on double copy integration;

[0045] (3) The application of the system described in the first aspect of the present invention or the ROSA26 site Donor vector and / or H11 site Donor vector described in the second aspect of the present invention in constructing a transgenic animal bioreactor for producing therapeutic recombinant monoclonal antibodies.

[0046] Furthermore, the target antibody is a monoclonal antibody against Staphylococcus aureus enterotoxin B, and the antigen is Staphylococcus aureus enterotoxin B.

[0047] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0048] This invention provides, for the first time, a transgenic animal antibody high-yield expression system based on dual-copy integration and its related applications. By targeting and integrating dual-copy monoclonal antibody genes against Staphylococcus aureus enterotoxin B (SEB) at the safe sites of ROSA26 and H11 in mice, this invention achieves high systemic expression of the antibody in mice, significantly improving antibody yield and neutralizing activity. Furthermore, comparative experiments unexpectedly revealed that the concentration of LXY-Ab antibody in the serum of dual-copy mice increased more than 10-fold compared to single-copy mice. This result is a technical effect that would not have been anticipated by those skilled in the art based on existing technology. This invention provides a new approach and strategy for the development of low-cost, high-efficiency therapeutic antibody bioreactors, possessing significant application value and promising prospects for industrial transformation. Attached Figure Description

[0049] Figure 1 Schematic diagram of Donor at ROSA26 site;

[0050] Figure 2 : Schematic diagram of Donor at H11 site;

[0051] Figure 3 : Results of PCR-identified positive mice;

[0052] Figure 4 : Sanger sequencing results;

[0053] Figure 5 : Expression results of LXY-Ab in serum;

[0054] Figure 6 : Graph showing the expression results of LXY-Ab in saliva and milk;

[0055] Figure 7 : Graph showing the results of affinity assay for LXY-Ab expressed in mice;

[0056] Figure 8 : Competitive ELISA detection of antibodies derived from transgenic mice;

[0057] Figure 9 : The in vivo protective effect of LXY-Ab expressed in mice. Detailed Implementation

[0058] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0059] Example 1: Selection of sgRNAs targeting mouse ROSA26 and H11 safety sites and design of Donor

[0060] To achieve targeted integration of the LXY-Ab expression cassette, the references designed two sgRNAs to target the first intron (sgRNA sequence: 5′-ACTCCAGTCTTTCTAGAAGA-TGG-3′ (SEQ ID NO:3)) and the H11 site (sgRNA sequence: 5′-GAACACTAGTGCACTTATCC-TGG-3′ (SEQ ID NO:4)) of the ROSA26 gene, respectively. The sgRNAs were prepared by chemical synthesis (GenScript synthesis).

[0061] Donor vector design: The mammalian highly expressed promoter CAG was selected, followed by a signal peptide-light chain-IRES element-signal peptide-heavy chain, and then a copy was repeated as the targeting vector. The left and right homologous arms are approximately 2 kb each (see the schematic diagrams of the ROSA26 site and H11 site donor vectors, respectively). Figure 1 and Figure 2 As shown), the Donor vector was synthesized by GenScript.

[0062] The nucleotide sequence of the Donor at the ROSA26 site is shown in SEQ ID NO:1, and the nucleotide sequence of the Donor at the H11 site is shown in SEQ ID NO:2.

[0063] The amino acid sequence of the heavy chain variable region of the antibody LXY-Ab is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region of the antibody LXY-Ab is shown in SEQ ID NO:14.

[0064] Example 2: Gene knock-in mice with safe sites of ROSA26 and H11 were prepared by injecting Cas9 protein, sgRNA and donor vector into mouse zygotes.

[0065] 1. Experimental Methods

[0066] (1) C57BL / 6J (females 3-4 weeks old; males 8-10 weeks old) fertilized eggs were obtained from the uterus of donor female mice 3.5 days after mating;

[0067] (2) Install the microinjection equipment and prepare the necessary reagents and materials;

[0068] (3) Select fertilized eggs with good morphology and appropriate development and transfer them to the culture medium in the injection dish;

[0069] (4) Prepare the RNP complex by mixing Cas9 protein, sgRNA as described in Example 1, and Donor with nuclease-free water containing 0.1 Mm EDTA. The final concentrations of the three components are as follows: Cas9: 30 ng / μL; sgRNA: 5 ng / μL; Donor: 10 ng / μL. Incubate the RNP to be injected in a 37°C incubator for 15 minutes before injection.

[0070] (5) The RNP to be injected is drawn into the injection needle and injected into the pronucleus of the fertilized egg one by one. The pronucleus of the successfully injected fertilized egg will be significantly enlarged.

[0071] (6) The injected fertilized eggs were cultured in vitro in M16 medium (SIGMA; M7292) to the 2-cell stage; the culture conditions were 37℃ and 5% CO2.

[0072] (7) Select 8-10 week old female mice that are in estrus naturally and put them in the same cage as neutered male mice to obtain surrogate female mice;

[0073] (8) The RNP-injected fertilized eggs were transferred into the uterus of a surrogate mother mouse;

[0074] (9) Place the surrogate mother mouse in a clean cage and keep it warm until it wakes up, then put it back in the cage for feeding.

[0075] (10) The fertilized egg transfer is completed, and the mice are born;

[0076] (11) When the mice are about 1 week old, cut off their toes and send them for PCR testing. They are then numbered and separated into different cages after 3 weeks.

[0077] (12) Genomic DNA extraction, lysis buffer of Triton X-100 and protease mixture, lyse sample overnight;

[0078] (13) The next day, the protease was inactivated by high temperature, and the supernatant was collected by centrifugation and used as a PCR template;

[0079] (14) PCR amplification and electrophoresis detection;

[0080] (15) Gel recovery of PCR fragments and sequencing identification;

[0081] (16) Analyze the sequencing results and record the positive mouse numbers.

[0082] Positive gene knock-in mice were obtained using PCR detection and TA cloning sequencing. The PCR identification primers are shown in Table 1 below.

[0083] Table 1 PCR identification primers

[0084]

[0085] 2. Experimental Methods

[0086] The results are as follows Figure 3 and Figure 4 As shown, PCR electrophoresis bands indicated that mice 1, 2, 3, 4, and 5 targeting ROSA26 and mice targeting H11 were all positive. Figure 3 The 5' and 3' Sanger sequencing results also showed that Donor successfully inserted into the corresponding site ( Figure 4 ).

[0087] Example 3: Detection of LXY-Ab expression in positive mice

[0088] 1. Experimental Methods

[0089] The expression of LXY-Ab in the positive mice obtained in Example 2 was detected using ELISA. Serum, saliva, and milk were collected from genotype-positive mice. LXY-Ab expression was detected in all three bodily fluids of the transgenic mice. The specific steps are as follows:

[0090] (1) Add 100 µL of the predetermined concentration of GAH (Goat anti-human IgG) capture antibody (1 µg / mL) to each well of a 96-well microplate. Place the plate at 4°C overnight for coating.

[0091] (2) Seal the wells with blocking solution (4% skim milk powder solution, 0.05% Tween-20 PBST) to avoid nonspecific binding. Incubate at room temperature for 1 hour, then discard the liquid and wash gently 3 times (200 µL of washing solution each time).

[0092] (3) Samples: Serially dilute mouse serum samples of the concentration to be tested into each well, adding 100 µL. Incubate at room temperature for 1 hour to allow the antibodies in the serum to bind to the capture antibodies. Standards: Use a series of concentrations (starting at 10 μg / mL, diluted 1:5) of the commercially available antibody cetuximab (Merck) as standards, and plot a standard curve based on OD values.

[0093] (4) After removing the serum sample, wash three times with PBS containing 0.05% Tween-20, 200 µL each time, to ensure the removal of non-specific conjugates.

[0094] (5) Select a suitable secondary antibody (such as HRP-labeled anti-human IgG secondary antibody). Dilute the secondary antibody 1:3000 and add 100 µL to each well. Incubate at 37°C for 30 min.

[0095] (6) After removing the secondary antibody, wash three times with PBS containing 0.05% Tween-20, 200 µL each time.

[0096] (7) Add TMB substrate solution (100 µL / well) and incubate in the dark for 10-30 minutes until the color appears.

[0097] (8) The reaction was terminated by using 2 M sulfuric acid (100 µL / well) to stop the substrate reaction.

[0098] (9) Use an ELISA reader to read the absorbance (OD value) of each well at a wavelength of 450 nm.

[0099] 2. Experimental Results

[0100] The results are as follows Figure 5 and Figure 6As shown, the above results demonstrate that the CRISPR / Cas9-mediated "safe site" site-directed integration systemic continuous expression bioreactor can efficiently and effectively express the target protein required by humans. Compared with single-copy mice (Rosa26[KI / +]: 0.93 mg / mL, Rosa26[KI / KI]: 2.58 mg / mL, H11[KI / +]: 1.35 mg / mL, H11[KI / KI]: 2.35 mg / mL, where [KI / +] indicates heterozygous and [KI / KI] indicates homozygous. The preparation method of single-copy mice is the same as described in Example 2, the only difference being single-copy instead of double-copy), the concentration of LXY-Ab in the serum of double-copy mice prepared in this invention is increased by more than 10 times (Rosa26[KI / +]-2: 24.71 mg / mL, Rosa26[KI / KI]-2: 34.22 mg / mL, H11[KI / +]-2: 28.78 mg / mL, H11[KI / KI]-2: 38.16 mg / mL, where [KI / +] indicates heterozygous and [KI / KI] indicates homozygous) Figure 5 This unexpected discovery in the present invention represents a technical effect that would not have been anticipated by those skilled in the art based on existing technology, as antibodies are also secreted in milk and saliva. Figure 6 ).

[0101] Example 4: Detection of LXY-Ab binding activity in the serum of transgenic mice

[0102] 1. The affinity constants between LXY-Ab and antigen in serum were detected using the Biacore T200.

[0103] The binding kinetics of LXY-Ab were determined using a Biacore T200 instrument (Cytiva) at 25°C in HBS-EP buffer (BR100669, Cytiva). LXY-R26, LXY-H11, and LXY-CHO refer to LXY-Ab expressed in the serum of mice knocked in at the ROSA26 site, LXY-Ab expressed in the serum of mice knocked in at the H11 site, and LXY-Ab expressed in CHO, respectively. First, the carboxyl groups on the CM5 sensor chip matrix were activated using a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form an active ester that reacts with amino groups. Subsequently, the SEB antigen was dissolved at a concentration of 1 μg / mL in 10 mM sodium acetate buffer at pH 5.0 and covalently coupled to the chip surface, immobilizing approximately 180 response units (RU). The unreacted reactive ester residues were then blocked with ethanolamine.

[0104] Binding kinetics were performed using a multi-cycle kinetics method, with LXY-Ab diluted in a two-fold gradient across a concentration range of 3.125 to 50 nM. Each binding cycle lasted 180 seconds, and each dissociation cycle lasted 300 seconds, at a flow rate of 30 μL / min, followed by chip surface regeneration. Finally, the binding kinetic parameters were globally calculated using Biacore T200 Evaluation Software 3.1 (Cytiva) based on a one-to-one Langmuir binding model.

[0105] Biacore experimental results showed that, compared with the LXY-Ab monoclonal antibody expressed in CHO, the antibody affinity expressed in mice was: LXY-R26 (ROSA26 safe site transgenic LXY-Ab mice) KD = 1.08 × 10⁻⁶. -10 M, LXY-H11 (H11 safe site transgenic LXY-Ab mice) KD=1.54×10 -10 M was lower than that of the antibody (LXY-CHO) expressed on CHO cells (KD = 1.80 × 10⁻⁶). -10 M indicates that the antibodies obtained from transgenic mice have a higher affinity for the antigen SEB. Figure 7 ).

[0106] 2. Competitive ELISA detection of whether antibodies in the serum of transgenic mice and antibodies expressed in CHO cells recognize the same epitopes.

[0107] Coat the antigen; dilute the purified antigen (e.g., SEB protein) at an appropriate concentration (e.g., 1-5 μg / mL) with carbonate buffer (pH 9.6). Add 100 μL to each well and incubate overnight at 4°C or for 2 hours at 37°C.

[0108] Blocking: Block nonspecific binding sites with 5% skim milk powder or 1% BSA solution (prepared with PBS). Add 200 μL to each well and incubate at room temperature for 1 hour.

[0109] Competitive reaction: Add 100 μL of serially diluted serum (LXY-R26 or LXY-H11) and biotinylated antibody (LXY-CHO) mixture to each well of the sealed plate and incubate at 37°C for 1 hour.

[0110] Wash the plate 3-5 times with PBST (0.05% Tween-20 PBS) to remove unbound components.

[0111] Add enzyme-labeled secondary antibody (such as HRP-conjugated streptavidin) at a rate of 100 μL per well and incubate at 37°C for 30-60 minutes.

[0112] Wash the plate; wash the plate 3-5 times with PBST.

[0113] Substrate color development: Add 100 μL of TMB color development solution and incubate at room temperature in the dark for 10-20 minutes, adjusting the time according to the color development effect.

[0114] To terminate the reaction, add 50 μL of 2 M H2SO4.

[0115] Detect absorbance and read the OD value at 450 nm.

[0116] Competitive ELISA results showed that both LXY-R26 and LXY-H11, derived from transgenic mice, specifically competed with antibodies expressed in CHO cells, indicating that the epitopes of the antibodies expressed from transgenic mice did not drift and were consistent with the maternal antibody LXY-CHO. Figure 8 ).

[0117] Example 5: Detection of the in vivo neutralizing activity of LXY-Ab in the serum of transgenic mice

[0118] 1. Experimental Methods

[0119] Female BALB / c mice aged 6-8 weeks were sensitized via intraperitoneal injection of D-galactosamine hydrochloride (D-GalN, 1 g / kg; Sigma-Aldrich, catalog number G0500). Thirty minutes after sensitization, mice were intraperitoneally injected with either SEB (0.25 mg / kg, dissolved in PBS) or PBS as a control. To assess treatment efficacy, another group of mice was injected intraperitoneally with D-GalN (1 g / kg), followed 30 minutes later by a mixture of pre-mixed SEB (0.25 mg / kg) and LXY-Ab transgenic mouse serum (containing LXY-Ab at a dose of 25 or 100 mg / kg) or purified LXY-CHO antibody (100 mg / kg). Mice survival was monitored for 120 hours post-injection, and mortality was calculated at the end of the experiment. Statistical analysis was performed using Kaplan-Meier survival curves and log-rank tests, using GraphPad Prism v9.0 software.

[0120] 2. Experimental Results

[0121] Post-challenge treatment results showed that the LXY-Ab antibodies (LXY-R26 or LXY-H11) expressed in mice exhibited better neutralizing activity than the maternal antibodies. Figure 9 This result is also a technical effect that would not have been anticipated by those skilled in the art based on existing technology.

Claims

1. A transgenic animal antibody high-yield expression system based on dual-copy integration, characterized in that, The system includes targeted integration of a target antibody expression cassette into the ROSA26 safe site and / or H11 safe site in mammals, respectively. The target antibody expression cassette contains a mammalian high-expression promoter CAG, a signal peptide-the target antibody light chain-IRES element-signal peptide-the target antibody heavy chain connected in sequence, and the signal peptide-the target antibody light chain-IRES element-signal peptide-the target antibody heavy chain repeats one copy to form a double-copy structure.

2. The system according to claim 1, characterized in that, The mammalian high-expression promoter CAG also has a Kozak sequence downstream, and the target antibody expression cassette has a β-globin polyadenylate signal at its end; Optionally, the targeted integration of the ROSA26 safe site is achieved via sgRNA with a sequence as shown in SEQ ID NO:3; Optionally, the targeted integration of the H11 safe site is achieved via sgRNA with a sequence as shown in SEQ ID NO:

4.

3. The system according to claim 1, characterized in that, The target antibody is a monoclonal antibody against Staphylococcus aureus enterotoxin B; Optionally, the amino acid sequence of the heavy chain variable region of the monoclonal antibody against Staphylococcus aureus enterotoxin B is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

14. Optionally, the mammal is a mouse, rat, guinea pig, hamster, pig, cow, sheep, horse, rabbit, or monkey.

4. A Donor carrier assembly for constructing the system according to any one of claims 1-3, characterized in that, The Donor vector combination includes the ROSA26 site Donor vector and / or the H11 site Donor vector; The ROSA26 site Donor vector comprises, in sequence, a 5′ homologous arm, a CAG promoter, a Kozak sequence, a signal peptide-light chain variable region VK-human light chain constant region hIgK-CL-IRES2 element-signal peptide-heavy chain variable region VH-human heavy chain constant region hlgG1-CH, a signal peptide-light chain variable region VK-human light chain constant region hIgK-CL-IRES2 element-signal peptide-heavy chain variable region VH-human heavy chain constant region hlgG1-CH, a β-globin poly (A) signal, and a 3′ homologous arm; The H11 site Donor vector comprises, in sequence, a 5′ homologous arm, a CAG promoter, a Kozak sequence, a signal peptide-light chain variable region VK-human light chain constant region hIgK-CL-IRES2 element-signal peptide-heavy chain variable region VH-human heavy chain constant region hlgG1-CH, a signal peptide-light chain variable region VK-human light chain constant region hIgK-CL-IRES2 element-signal peptide-heavy chain variable region VH-human heavy chain constant region hlgG1-CH, a β-globin poly (A) signal, and a 3′ homologous arm.

5. The Donor carrier assembly according to claim 4, characterized in that, The nucleotide sequence of the ROSA26 site Donor vector is shown in SEQ ID NO:1; The nucleotide sequence of the Donor vector at the H11 site is shown in SEQ ID NO:

2.

6. A method for constructing the system according to any one of claims 1-3, characterized in that, The method includes the following steps: (1) Design and synthesize sgRNA targeting the ROSA26 safe site and / or sgRNA targeting the H11 safe site; (2) Construct the ROSA26 site Donor vector and / or H11 site Donor vector as described in claim 4 or 5; (3) Obtain fertilized eggs from the uterus of the donor female mouse; (4) Mix Cas9 protein, sgRNA targeting the ROSA26 safe site and / or sgRNA targeting the H11 safe site, ROSA26 site Donor vector and / or H11 site Donor vector to form an injection mixture, dilute it and draw it into the injection needle, and inject it into the nucleus and cytoplasm of the fertilized egg one by one. (5) The injected fertilized eggs were cultured in vitro to the 2-cell stage; (6) Select a suitable female mouse and a vasectomized male mouse to obtain a surrogate female mouse, and transplant the fertilized eggs cultured to the 2-cell stage into the uterus of the surrogate female mouse; (7) After the surrogate mother mouse gives birth, the genomic DNA of the 1-week-old mice is extracted by cutting off the toes. The positive mice obtained are the high-yield expression system carrying the target antibody. Optionally, in step (4), the concentration of Cas9 protein in the injection mixture is 10-50 ng / μL, the concentrations of sgRNA targeting the ROSA26 safe site and the sgRNA targeting the H11 safe site are 2-7 ng / μL and 2-7 ng / μL, respectively, and the concentrations of the ROSA26 site Donor vector and the H11 site Donor vector are 5-15 ng / μL and 5-15 ng / μL, respectively. Optionally, in step (4), the concentration of Cas9 protein in the injection mixture is 30 ng / μL, the concentrations of sgRNA targeting the ROSA26 safe site and the sgRNA targeting the H11 safe site are 5 ng / μL and 5 ng / μL, respectively, and the concentrations of the ROSA26 site Donor vector and the H11 site Donor vector are 10 ng / μL and 10 ng / μL, respectively.

7. The method according to claim 6, characterized in that, In step (5), the conditions for in vitro culture of fertilized eggs are 37°C, 5% CO2, and M16 medium.

8. A transgenic animal bioreactor, characterized in that, The transgenic animal bioreactor is a transgenic animal bioreactor prepared by the method described in claim 6 or 7.

9. Applied to any of the following aspects: (1) The use of the system of any one of claims 1-3 or the ROSA26 site Donor vector and / or H11 site Donor vector as described in claims 4 or 5 in the preparation of a therapeutic drug for antigen-related diseases corresponding to the target antibody; (2) The application of the system of any one of claims 1-3 or the ROSA26 site Donor vector and / or H11 site Donor vector as described in claims 4 or 5 in constructing a transgenic animal antibody high-yield expression system based on double-copy integration; (3) The use of the system of any one of claims 1-3 or the ROSA26 site Donor vector and / or H11 site Donor vector as described in claims 4 or 5 in constructing a transgenic animal bioreactor for producing therapeutic recombinant monoclonal antibodies.

10. The application according to claim 9, characterized in that, The target antibody is a monoclonal antibody against Staphylococcus aureus enterotoxin B, and the antigen is Staphylococcus aureus enterotoxin B.

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