A method for constructing an immunized animal model for preparing a biofusion enzyme antibody and application thereof

Abstract

This invention discloses a method for constructing an immune animal model for preparing biofusion enzyme antibodies and its application. The method includes the following steps: designing and screening sgRNAs with high cleavage efficiency based on signal protein genes, and constructing an sgRNA-Cas9 expression vector; linearizing the plasmid by double enzyme digestion, and then ligating it with a signal protein gene fragment containing left and right homologous arms to obtain the Donor plasmid; co-transfecting the sgRNA-Cas9 expression vector and the Donor plasmid into target animal somatic cells, and screening to obtain positive somatic cells that stably integrate the target gene; using the positive somatic cells as nuclear donors for nuclear transfer to construct recombinant embryos, and transferring the recombinant embryos into recipient female animals; after delivery, identifying transgenic animal individuals carrying biofusion enzyme antibodies by genomic PCR. Based on this transgenic animal model, different target antibodies with clinical value can be developed.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for constructing an immune animal model for preparing biological fusion enzyme antibodies and its application. Background Technology

[0002] As is well known, antibodies recognize antigens through their binding sites, interacting with specific epitopes on the antigens they bind to. This specific antibody-antigen interaction has wide applications, including antigen detection and clinical diagnosis. To detect antibody-antigen interactions, antibodies or antigens are typically labeled with signal-generating molecules. These molecules include small chemicals capable of generating light, electrical, sound, or magnetic signals, and reporter proteins capable of similarly generating these detectable signals, primarily colorimetric, fluorescent, or bioluminescent light signals. The signal-generating chemicals or reporter proteins are usually coupled to the antibody or antigen via chemical reactions with specific types of amino acids at specific or non-specific sites. This conjugated product is indeterminate, its composition is heterogeneous, and the coupling sites on the antibody or antigen can also affect the antibody-antigen interaction and thus the immunoassay. Due to these factors, the inherent limitations of chemical coupling are particularly evident in different immunological detection platforms.

[0003] (i) This makes it difficult to directly link the signal enzyme to an antibody (commonly known as a primary antibody) targeting a specific target in immunohistochemical experiments. First, the coupling efficiency is difficult to control, resulting in significant batch-to-batch differences in antibodies. Second, the coupling yield is very low, currently around 30-60%, causing a great waste of antibodies. These insurmountable technical barriers have greatly hindered the development of immunohistochemical technology towards a faster and more accurate direction.

[0004] (ii) In chemiluminescence and enzyme-linked immunosorbent assay (ELISA) platforms, in the detection modes of double antibody sandwich detection of proteins and labeled antibody competitive detection of small molecules, the strong randomness, low controllability and low coupling yield of antibody chemical coupling will lead to poor batch-to-batch controllability of the kit.

[0005] With the development of molecular biology techniques, especially the emergence of novel gene-editing technologies like CRISPR / Cas9, they have been widely applied in many fields such as basic research, biotechnology, and biomedicine. The core of the CRISPR / Cas9 system is sgRNA and the Cas9 protein. Cas9 is a nuclease that recognizes and cuts target DNA based on the guide sequence of sgRNA. sgRNA is composed of crRNA and tracrRNA, where crRNA is responsible for recognizing the target sequence, and tracrRNA binds to Cas9 and assists in its localization and cleavage of the target DNA. This mechanism enables CRISPR / Cas9 to achieve highly specific gene editing. Transgenic animals can be created by microinjecting exogenous DNA into the pronucleus of a fertilized egg, which is then implanted into the oviduct of a recipient female in estrus synchronization. Some transgenic animals can also be created using embryonic stem cells by microinjecting transfected embryonic stem cells into embryos in the blastocyst stage, followed by implantation of the embryo into the oviduct of a recipient female in estrus synchronization, and then transfecting cells with exogenous DNA. Theoretically, proteins encoded by exogenous genes will be produced in transgenic animals, either as standalone proteins or fused with endogenous proteins according to their original design. When a DNA-encoding signaling protein is selectively inserted into one end of the gene that produces the immunoglobulin, the transgenic animal will produce signaling protein fusion antibodies. These antibodies are fully recognized homogeneous molecules without any other effects. When the signaling protein gene is fused to one end of the immunoglobulin constant region gene, it is expected that the transgenic animal will produce a highly diverse library of antigen-specific signaling protein fusion antibodies after immunization. By screening for these signaling protein fusion antibodies, suitable antibodies for immunoassay can be identified. Transgenic animals modified in this way serve as a key platform for solving the aforementioned biological fusion antibody technology. Based on this transgenic animal platform, combined with the application of molecular biology techniques, it is possible to fuse different signaling proteases with antibodies for expression, better serving various immunoassay platforms. Compared to chemically conjugated antibodies, protease fusion antibodies produced using such a technology platform have many advantages, including: (1) compared to heterogeneous chemically conjugated antibodies, it is a molecule that is completely secreted along with the antibody, without any heterogeneous reaction; (2) it has strict signal / antibody stoichiometry, allowing for good control of signal intensity; and (3) in immunohistochemical platforms, it allows bypassing the need to amplify the signal with conventionally labeled secondary antibodies, thus simplifying the immunoassay procedure. Currently, there is no such transgenic animal platform to achieve this. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing an immune animal model for preparing biological fusion enzyme antibodies, thereby constructing a transgenic animal model that can secrete antibodies of fusion signal proteins. Based on this transgenic animal model, this invention has developed several target antibodies with clinical application value, and has conducted preliminary performance verification on different immune detection platforms based on the clinical application characteristics of different target antibodies.

[0007] The technical solution adopted in this invention is as follows:

[0008] A method for constructing an immune animal model for preparing biological fusion enzyme antibodies, comprising the following steps:

[0009] S1) Design and screen sgRNAs for the CRISPR / Cas9 system: Based on the signal protein gene, design and screen sgRNAs with high cleavage efficiency, and construct sgRNA-Cas9 expression vectors;

[0010] S2) Constructing the Donor plasmid: After linearizing the plasmid by double enzyme digestion, ligate it with a signal protein gene fragment containing left and right homologous arms to obtain the Donor plasmid;

[0011] S3) Preparation of transgenic animal individuals: The sgRNA-Cas9 expression vector and Donor plasmid are co-transfected into the target animal somatic cells, and positive somatic cells that stably integrate the target gene are screened to obtain them; the positive somatic cells are used as nuclear donors for nuclear transfer to construct recombinant embryos, and the recombinant embryos are transferred into the recipient female animal. After delivery, the transgenic animal individuals carrying biological fusion enzyme antibodies are identified by genomic PCR.

[0012] The sgRNA sequence includes any one of the following: GGAGCATGCAGCTTTTTCTT sequence as shown in SEQ ID NO. 25, AACGGCTCCACCACGCTCGG sequence as shown in SEQ ID NO. 59, and TGATGATTACCACGCTACGA sequence as shown in SEQ ID NO. 91.

[0013] In step S1), a variety of design schemes are systematically screened to obtain animal genome target sites suitable for knocking in the target signal protein gene. Then, specific sgRNAs and their corresponding amplification primers are designed to construct the sgRNA expression plasmids required for the CRISPR / Cas9 system. Subsequently, the cleavage efficiency of each sgRNA is verified by in vitro cleavage experiments, and the sgRNA with the highest cleavage activity is screened out.

[0014] The sgRNA with the best cleavage efficiency was initially screened, off-target sites were identified, and off-target detection primers were designed to perform off-target detection to confirm the accuracy of the transferred gene.

[0015] Specific sgRNAs and their corresponding amplification primers were designed and screened for the insertion sites of the Safe Harbor gene in different animals to construct the sgRNA expression plasmids required for the CRISPR / Cas9 system.

[0016] For different signal protein tags and different animals, the sgRNA, Donor plasmid, and specific insertion site vary. In the construction method, this invention fully considers the structure of the inserted protein and its conserved genomic location in the animal, determining the sgRNA design and screening, the Donor plasmid design process, and the selection of the fusion site based on these factors. The screening method for SafeHarbor gene insertion sites in different animals is as follows:

[0017] 1.1 Obtain the complete genome sequence of the target immunized animal, and use bioinformatics tools (such as TargetFinder, PrimateAI-3D, etc.) to search for potential neutral insertion sites in non-functional coding regions and non-critical regulatory regions of the genome. Sites located in exons, core promoters, enhancers and microRNA binding regions of coding genes are excluded, and a preliminary set of candidate Safe Harbor sites is obtained.

[0018] 1.2 Safety and stability verification screening: For candidate Safe Harbor sites, bioinformatics algorithms are used to predict the effect of foreign gene insertion on the expression of surrounding genes, and sites that may cause gene silencing or abnormal activation are eliminated; conserved sites that have been verified to have safety in the same species are retained.

[0019] 1.3 Feasibility and Expression Efficiency Screening: The feasibility of homologous recombination at candidate sites was tested, and sites with high sequence conservation and easy design of homologous arms were selected. Through in vitro cell experiments, reporter genes (such as mCherry) were initially inserted into candidate sites, and the expression level and stability of reporter genes were tested. Finally, Safe Harbor gene insertion sites that "do not affect the normal expression of the animal's own genes after insertion of the target gene and can be stably and efficiently expressed" were selected.

[0020] In step S2), the plasmid is selected from the FXpcDNA3.1(+) vector plasmid modified with special elements in our laboratory. Original primers for amplification of the left and right homologous arms of the target signal protein gene are designed, and the Donor plasmid containing the target gene is constructed through an optimized program.

[0021] The FXpcDNA3.1(+) vector plasmid modified by our laboratory with special elements is a traditional pcDNA3.1(+) vector with the addition of highly efficient enhancers and stable expression elements, so as to achieve efficient and stable integration of foreign genes into the genome of transgenic animals.

[0022] The animals mentioned can be rats, dogs, rabbits, pigs, guinea pigs, donkeys, sheep, goats, chickens, cattle, llamas, or camels, etc.

[0023] The signaling proteins refer to proteins with biological activities that can indicate signals, including but not limited to green fluorescent protein (GFP) and its variants, resonance energy transfer (RET) donor molecules, RET acceptor molecules, protein fragment complementation analysis (PCA) bait proteins, PCA prey proteins, horseradish peroxidase (HRP), alkaline phosphatase (AP), luciferase, including but not limited to firefly luciferase, reninase, deep-sea shrimp luciferase and its variants, p-galactosidase, chloramphenicol acetyltransferase, glucose oxidase, acetate kinase, xanthine oxidase, or glucose-6-phosphate dehydrogenase, etc.

[0024] Signal fusion proteins are knocked into possible fusion sites on antibodies, including the N-terminus of the antibody heavy chain (after the ATG start codon) and the C-terminus (before the stop codon), and the N-terminus of the antibody light chain (after the ATG start codon) and the C-terminus (before the stop codon). Monomeric IgG is used as an example. Other types of antibodies may form different complexes, such as IgA dimers and IgM pentamers. Signal fusion proteins can form different complexes depending on the antibody type. In all cases, the signal fusion protein should retain its antigen-binding ability and be able to generate a signal to aid in subsequent detection. This invention uses knocking into the N-terminus of the monomeric IgG light chain (after the ATG start codon and before the stop codon) as an example for illustration.

[0025] A CD5 antibody carrying the signal protein HRP, wherein the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 3 or a sequence having more than 80% sequence similarity to SEQ ID NO: 3; and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1 or a sequence having more than 80% sequence similarity to SEQ ID NO: 1.

[0026] The nucleotide sequence encoding the CD5 antibody carrying the signal protein HRP.

[0027] The TSH antibody Mab008 is tagged with horseradish peroxidase (HRP) signaling protein, wherein the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO.15 or a sequence having more than 80% sequence similarity to SEQ ID NO.15; and the amino acid sequence of the heavy chain variable region is SEQ ID NO.13 or a sequence having more than 80% sequence similarity to SEQ ID NO.13.

[0028] The TSH antibody Mab007 is tagged with horseradish peroxidase (HRP) signaling protein, wherein the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO.11 or a sequence having more than 80% sequence similarity to SEQ ID NO.11; and the amino acid sequence of the heavy chain variable region is SEQ ID NO.9 or a sequence having more than 80% sequence similarity to SEQ ID NO.9.

[0029] The TSH antibody Mab005 is tagged with horseradish peroxidase (HRP) signaling protein, wherein the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO.7 or a sequence having more than 80% sequence similarity to SEQ ID NO.7; and the amino acid sequence of the heavy chain variable region is SEQ ID NO.5 or a sequence having more than 80% sequence similarity to SEQ ID NO.5.

[0030] The nucleotide sequences encoding the TSH antibodies Mab005, Mab007, and Mab008, which are tagged with the horseradish peroxidase (HRP) signaling protein.

[0031] The TSH antibodies Mab005, Mab007, and Mab008, which are tagged with the horseradish peroxidase (HRP) signaling protein, exhibit high specificity and sensitivity.

[0032] The present invention provides CD5 and TSH protein sequences containing specific antigenic epitopes for use in immunization experiments. The methods for obtaining CD5 and TSH proteins containing these specific antigenic epitope sequences include, but are not limited to, obtaining prokaryotic expression proteins (E. coli system), eukaryotic expression proteins (yeast system, insect cell system, CHO cell system, HEK29 cell system), and synthesized polypeptide fragments through genetic engineering. The present invention uses CD5 synthetic polypeptide fragments and TSH recombinant expression proteins as examples for illustration.

[0033] This invention screens for highly specific and sensitive antibodies against CD5 and TSH proteins tagged with biofusion enzymes. These antibodies include polyclonal antibodies, monoclonal antibodies, Fab and its single-chain Fv (scFv) fragments, bispecific antibodies, heteroconjugates, and human and humanized antibodies. These antibodies can be obtained through various screening methods, including traditional cell fusion, phage library display, and single-B cell screening. They can be produced in various ways, including hybridoma culture, recombinant expression in bacterial or mammalian cell culture, and recombinant expression in transgenic animals. The choice of antibody production method depends on several factors, including the desired antibody structure, the importance of the carbohydrate moiety to the antibody, ease of culture and purification, and cost. Many different antibody structures can be generated using recognized standard expression techniques, including full-length antibodies, antibody fragments (such as Fab and Fv fragments), and chimeric antibodies composed of different species. This invention illustrates the production of antibodies using a single-B cell screened monoclonal antibody and recombinant expression in mammalian cell culture as an example.

[0034] The CD5 and TSH proteins, which are highly expressed by biological fusion enzyme tags with high specificity and sensitivity, were used to validate clinical immunological experiments. These validations included, but were not limited to, experiments based on the immunological principle of antigen-antibody binding, such as immunohistochemistry (IHC), Western blot (WB), chemiluminescence immunoassay (CLIA), and enzyme-linked immunosorbent assay (ELISA). This invention uses immunohistochemistry (IHC), chemiluminescence immunoassay (CLIA), and enzyme-linked immunosorbent assay (ELISA) as examples for illustration.

[0035] Beneficial effects:

[0036] This invention provides a method for constructing an immune animal model for preparing biofusion enzyme antibodies, thereby constructing a transgenic animal model that can self-secrete fusion signal proteins. Based on this transgenic animal model, this invention has developed several target antibodies with clinical application value, and conducted preliminary performance verification on different immunoassay platforms based on the clinical application characteristics of different target antibodies. The obtained antibodies have highly expressed biofusion enzyme tags with strong specificity and high sensitivity. Attached Figure Description

[0037] Figure 1 PCR test results of F0 generation rabbits, where the bands from left to right are F001, F002, F003, F004, F005, F006, F007, F008, F009, Marker, Negative, Positive;

[0038] Figure 2 F0 generation rabbit TMB color development results;

[0039] Figure 3 PCR test results of F1 generation rabbits, where the bands from left to right are M, negative, positive, M, F101, F102, F103, F104, F105, F106, F107, F108, F109, F110, F111, F112, F113, F114, F115, F116, F117, F118;

[0040] Figure 4 Flowchart for constructing transgenic rabbits tagged with horseradish peroxidase (HRP) signaling protein using CRISPR / Cas9 technology;

[0041] Figure 5 CD5 protein structure diagram;

[0042] Figure 6 Comparison of the detection results of different biological fusion enzyme CD5 antibodies and 4C7 in the same peripheral T-cell lymphoma tissue: A. 4C7 antibody staining result of peripheral T-cell lymphoma tissue-paraffin section (positive control); B. CD5-Mab001 staining result of peripheral T-cell lymphoma tissue-paraffin section (weak); C. CD5-Mab002 staining result of peripheral T-cell lymphoma tissue-paraffin section (strong); D. CD5-Mab003 staining result of peripheral T-cell lymphoma tissue-paraffin section (weak); E. CD5-Mab004 staining result of peripheral T-cell lymphoma tissue-paraffin section (weak); F. CD5-Mab005 staining result of peripheral T-cell lymphoma tissue-paraffin section (strong).

[0043] Figure 7 Comparison of test results for CD5-Mab002 (clone number PM19A9) antibody and 4C7 antibody against different samples. A: Tissue 1 - tonsil - paraffin section CD5-Mab002 (clone number PM19A9) antibody staining results; B: Tissue 1 - tonsil - paraffin section 4C7 antibody staining results (positive control); C: CD5-Mab002 (clone number PM19A9) antibody staining results; D: 4C7 antibody staining results (positive control); E: CD5-Mab002 (clone number PM19A9) antibody staining results; F: 4C7 antibody staining results (positive control).

[0044] Figure 8 This invention provides electrophoresis images of the constructed TSH recombinant plasmid and SDS-PAGE images of the expressed recombinant TSH protein;

[0045] Figure 9 Standard curve fitting plot of TSH-Mab005 antibody fusion enzyme (chemiluminescence platform).

[0046] Figure 10 Standard curve fitting plot of TSH-Mab007 antibody fusion enzyme (chemiluminescence platform);

[0047] Figure 11 Standard curve fitting plot of TSH-Mab008 antibody fusion enzyme (chemiluminescence platform). Detailed Implementation

[0048] The FXpcDNA3.1(+) vector plasmid is a modification of the pcDNA3.1(+) vector plasmid designed to promote the stable integration and sustained expression of exogenous genes. Specifically, it integrates the interleukin-2 (IL-2) signal peptide sequence and the Epstein-Barr virus nuclear antigen 1 (EBNA1) stable expression element into its basic backbone.

[0049] The following description uses horseradish peroxidase as a signaling protein, which is merely exemplary and not limiting. Those skilled in the art, based on the disclosed technical concept and solution, can understand and anticipate that replacing horseradish peroxidase with other signaling proteins or their functional equivalents will achieve the objectives of the present invention and obtain the corresponding technical effects without requiring inventive effort.

[0050] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] The following examples use mice, rabbits, and alpacas as examples to illustrate the construction method of transgenic animal models. They are only used to explain the present invention and not to limit the present invention.

[0052] A method for constructing a transgenic rabbit model tagged with horseradish peroxidase (HRP) signaling protein includes the following steps:

[0053] 1. Through systematic screening of various design schemes, animal genomic target sites suitable for horseradish peroxidase (HRP) signal protein gene knock-in were successfully identified, and specific sgRNAs and their corresponding amplification primers were designed to construct the sgRNA expression plasmids required for the CRISPR / Cas9 system. Subsequently, the cleavage efficiency of each sgRNA was verified through in vitro cleavage experiments, and the sgRNA with the highest cleavage activity was screened out. The finally determined sgRNA is: GGAGCATGCAGCTTTTTCTT (PAM sequence GGG).

[0054] The determined sgRNA primers are: F: CACCggagcatgcagctttttctt

[0055] R:AAACaagaaaaagctgcatgctcc

[0056] 2. Using the FXpcDNA3.1(+) vector plasmid modified with special elements in our laboratory, we designed amplification primers for the left and right homologous arms of the horseradish peroxidase (HRP) signaling protein gene. We then constructed a Donor plasmid containing the horseradish peroxidase (HRP) signaling protein using an optimized program. The FXpcDNA3.1(+) vector plasmid used in our laboratory is a traditional pcDNA3.1(+) vector with the addition of highly efficient enhancers and stable expression elements to achieve efficient and stable integration of the foreign gene into the transgenic rabbit genome.

[0057] 3. Preparation of transgenic animal individuals: The confirmed sgRNA / Cas9 plasmid and Donor plasmid are co-transfected into the target animal somatic cells, and positive somatic cells that stably integrate the target gene are screened to obtain them; the positive somatic cells are used as nuclear donors for nuclear transfer to construct recombinant embryos, and the recombinant embryos are transferred into the recipient female animal. After delivery, the transgenic animal individuals carrying biological fusion enzyme antibodies are identified by genomic PCR.

[0058] Based on the sgRNA with the best cleavage efficiency identified in the preliminary screening, off-target sites were determined and off-target detection primers were designed to perform off-target detection and confirm the accuracy of the transferred gene.

[0059] The FXpcDNA3.1(+) vector plasmid modifies the pcDNA3.1(+) vector plasmid by integrating the interleukin-2 (IL-2) signal peptide sequence and the Epstein-Barr virus nuclear antigen 1 (EBNA1) stable expression element into its basic backbone, aiming to promote the stable integration and continuous expression of foreign genes.

[0060] The amplification primers for the left and right homologous arms are:

[0061] sgRNA-HA-LF: AATT tataagacttttaaaagtctgatgg

[0062] sgRNA1-HA-RR: TCGA ttaacaagagtcagggcca.

[0063] The method for constructing a transgenic mouse model tagged with horseradish peroxidase (HRP) signaling protein includes the following steps:

[0064] 1. Through systematic screening of various design schemes, animal genomic target sites suitable for horseradish peroxidase (HRP) signal protein gene knock-in were successfully identified, and specific sgRNAs and their corresponding amplification primers were designed to construct the sgRNA expression plasmids required for the CRISPR / Cas9 system. Subsequently, the cleavage efficiency of each sgRNA was verified through in vitro cleavage experiments, and the sgRNA with the highest cleavage activity was screened out. The finally determined sgRNA is: AACGCTCCACCACGCTCGG (PAM sequence AGG).

[0065] The determined sgRNA primers are: F: CACCGacggctccaccacgctcgg

[0066] R:AAACccgagcgtggtggagccgtC

[0067] 2. Using the FXpcDNA3.1(+) vector plasmid modified with special elements in our laboratory, we designed original primers for the left and right homologous arms of the horseradish peroxidase (HRP) signaling protein gene. Through optimized programming, we constructed a Donor plasmid containing the horseradish peroxidase (HRP) signaling protein. The FXpcDNA3.1(+) vector plasmid used in our laboratory is a traditional pcDNA3.1(+) vector with added highly efficient enhancers and stable expression elements to achieve efficient and stable integration of the foreign gene into the transgenic mouse genome.

[0068] 3. Preparation of transgenic animal individuals: The confirmed sgRNA / Cas9 plasmid and Donor plasmid are co-transfected into the target animal somatic cells, and positive somatic cells that stably integrate the target gene are screened to obtain them; the positive somatic cells are used as nuclear donors for nuclear transfer to construct recombinant embryos, and the recombinant embryos are transferred into the recipient female animal. After delivery, the transgenic animal individuals carrying biological fusion enzyme antibodies are identified by genomic PCR.

[0069] Based on the sgRNA with the best cleavage efficiency identified in the preliminary screening, off-target sites were determined and off-target detection primers were designed to perform off-target detection and confirm the accuracy of the transferred gene.

[0070] The method for constructing a transgenic alpaca model tagged with horseradish peroxidase (HRP) signaling protein includes the following steps:

[0071] 1. Through systematic screening of various design schemes, animal genomic target sites suitable for horseradish peroxidase (HRP) signal protein gene knock-in were successfully identified, and specific sgRNAs and their corresponding amplification primers were designed to construct the sgRNA expression plasmids required for the CRISPR / Cas9 system. Subsequently, the cleavage efficiency of each sgRNA was verified through in vitro cleavage experiments, and the sgRNA with the highest cleavage activity was screened out. The finally determined sgRNA is: TGATGATTACCACGCTACGA (PAM sequence CGG).

[0072] The determined sgRNA primers are: F: CACCGgatgattaccacgctacga

[0073] R:AAACtcgtagcgtggtaatcatcC

[0074] 2. We selected the FXpcDNA3.1(+) vector plasmid, which has been modified with special elements in our laboratory, and designed original primers for amplifying the left and right homologous arms of the horseradish peroxidase (HRP) signal protein gene. We then constructed a Donor plasmid containing the horseradish peroxidase (HRP) signal protein using an optimized program. The FXpcDNA3.1(+) vector plasmid used in our laboratory is a traditional pcDNA3.1(+) vector with the addition of highly efficient enhancers and stable expression elements to achieve efficient and stable integration of the foreign gene into the transgenic alpaca genome. Specifically, we added the IL-2 signal peptide and the EBNA1 stable expression element to the pcDNA3.1(+) vector plasmid, which is beneficial for expression.

[0075] 3. Preparation of transgenic animal individuals: The confirmed sgRNA / Cas9 plasmid and Donor plasmid are co-transfected into the target animal somatic cells, and positive somatic cells that stably integrate the target gene are screened to obtain them; the positive somatic cells are used as nuclear donors for nuclear transfer to construct recombinant embryos, and the recombinant embryos are transferred into the recipient female animal. After delivery, the transgenic animal individuals carrying biological fusion enzyme antibodies are identified by genomic PCR.

[0076] Based on the sgRNA with the best cleavage efficiency identified in the preliminary screening, off-target sites were determined and off-target detection primers were designed to perform off-target detection and confirm the accuracy of the transferred gene.

[0077] Based on the transgenic animal model with antibodies that secrete biological fusion enzymes, protein targets for different clinical applications were selected and applied in the transgenic animal model, and related performance was verified.

[0078] Select the immunogen and administer immunization according to the following protocol, with the specific steps as follows:

[0079] Animal immunization and antiserum detection

[0080] 1) Select 3 animals in each group to construct the above-mentioned immunized animal model, mark and number them, collect 300~500ul of blood, and collect the pre-immunization serum by centrifugation after coagulation;

[0081] 2) First immunization: Calculate the required antigen volume based on an immunization dose of 300ug per animal, emulsify the antigen using an equal volume of Freund's complete adjuvant, and administer immunization via subcutaneous injection at multiple sites;

[0082] 3) Second immunization: Two weeks later, a second immunization is performed. The required antigen volume is calculated based on an immunization dose of 150ug per animal. An equal volume of Freund's incomplete adjuvant is used to emulsify the antigen, and multiple subcutaneous injections are used for immunization.

[0083] 4) Third immunization: Two weeks later, the third immunization is carried out. The required antigen volume is calculated based on an immunization dose of 150ug per animal. The antigen is emulsified using an equal volume of Freund's incomplete adjuvant and administered via subcutaneous injection at multiple points.

[0084] 5) Serum collection: One week after the third vaccination, 1000~2000ul of blood was collected intravenously, and the serum was collected by centrifugation after coagulation.

[0085] 6) ELISA is used to detect serum titers.

[0086] Based on the transgenic animal with secretory biological fusion enzyme antibody obtained by using rabbit as an example, protein targets for different clinical application scenarios were selected for application and related performance verification in this transgenic animal model to obtain antibodies with signal protein HRP.

[0087] Example 1: Constructing transgenic rabbits with horseradish peroxidase (HRP) signaling protein tags using CRISPR / Cas9 technology.

[0088] 1.1 Design of sgRNA for CRISPR / Cas9

[0089] Using horseradish peroxidase (HRP) as a signaling protein, the specific sequence was referenced from the gene sequence published by NCBI: PRXC1A. Based on the CRISPR / Cas9-sgRNA design website https: / / crispor.gi.ucsc.edu / , sgRNAs were designed for this gene, as shown in Table 1.

[0090] sgRNA1: GGAGCATGCAGCTTTTTCTT (PAM sequence GGG)

[0091] sgRNA2: TTCCTCAGAGAGCTTCGGCT (PAM sequence AGG)

[0092] sgRNA3: CGGCGGCTCCCGCTGATTGG (PAM sequence CGG)

[0093] The sgRNA was ligated into the lentiGuide-Puro (Addgene plasmid number 52963) vector to construct the sgRNA-Cas9 expression vector.

[0094] Table 1

[0095]

[0096] After the primer sequences were synthesized at the company, they were annealed, cooled to form double strands, and then ligated into the linearized plasmid lentiGuide-Puro, which had been digested with BsmBI and purified. The ligation status was subsequently confirmed by PCR and Sanger sequencing. The successfully ligated sgRNA plasmid vector was then amplified, and plasmid DNA was extracted.

[0097] 1.2 In vitro cleavage verification of sgRNA cleavage efficiency

[0098] (1) In vitro transcription of sgRNA

[0099] Table 2

[0100]

[0101] Using the sgRNA plasmid vector DNA as a template, PCR amplification was performed to obtain an in vitro transcription template for sgRNA. The annealing temperature was 60 °C and the extension time was 5 s. The amplification product was recovered, and in vitro transcription was performed according to the MEGAscript™ T7 transcription kit system and operating procedures. DNA was removed by adding DNase, and RNA was purified using the Vazyme RNA purification kit. The quality of the transcribed RNA was verified by agarose gel electrophoresis.

[0102] (2) Determining cutting efficiency by external cutting

[0103] Using rabbit genomic DNA as a template, the target sequence was amplified by PCR. Primer sequences are shown in Table 3. The annealing temperature was 57℃. The amplified product was recovered and used as a substrate to add Cas9 protein and transcripts for in vitro cleavage. After the cleavage reaction, agarose gel electrophoresis was used to determine band size, and image analysis of band grayscale was performed to determine cleavage efficiency. The sgRNA with the highest efficiency was selected. After final confirmation, sgRNA1 was selected as the most efficient in this study; therefore, only sgRNA1 was designed for subsequent off-target sites and detection primers.

[0104] Table 3

[0105]

[0106] The specific reaction conditions are shown in Table 4.

[0107] Table 4

[0108]

[0109] 1.2 Constructing the Donor plasmid

[0110] (1) Design homologous arm fragments and synthesize templates.

[0111] The donor plasmid used was FXpcDNA3.1(+) designed in our laboratory, containing the interleukin-2 (IL-2) signal peptide sequence and the Epstein-Barr nuclear antigen 1 (EBNA1) stable expression element. The plasmid was double-digested with ECORⅠ and XhoⅠ, and purified. Homologous arms were designed at both ends based on the restriction enzyme sites: the upstream sequence at ECORⅠ was used to create the left homologous arm (HA-L); the downstream sequence at XhoⅠ was used to create the right homologous arm (HA-R). Both homologous arms were added to the PRXC1A sequence, and the amplification template was synthesized by a company. The synthesized fragment included: HA-L 700bp, HA-R 501bp, with the PAM sequence replaced by GGC in the right homologous arm, and the PRXC1A sequence added in the middle.

[0112] The amplification primers here are:

[0113] sgRNA-HA-LF: AATT tataagacttttaaaagtctgatgg

[0114] sgRNA1-HA-RR:TCGAttaacaagagtcagggcca

[0115] (2) Carrier connection

[0116] The recovered and purified amplified fragments and linearized pcDNA™ 3.1(+) plasmid fragments were ligated using the enzyme ligation system shown in Table 5 below. The ligation was then preliminarily verified by transformation, picking of positive clones, and bacterial PCR, and further confirmed by Sanger sequencing.

[0117] Table 5

[0118]

[0119] 1.3 Co-transfection of rabbit somatic cells

[0120] sgRNA and Donor plasmids were co-transfected into rabbit somatic cells, and the transfection efficiency was determined by puromycin selection and fluorescence detection. Positive rabbit somatic cells with stable integration of the target gene were selected and used as nuclear transfer donor cells. Under a microscope, the nuclei of donor cells were injected into enucleated rabbit oocytes using micromanipulation techniques to construct recombinant embryos. Subsequently, the recombinant embryos were cultured in vitro to the morula or blastocyst stage. Well-developed embryos were selected and transferred to the oviduct or uterus of recipient rabbits undergoing estrus synchronization. After transfer, the recipient rabbits underwent regular ultrasound pregnancy monitoring to record embryo implantation and development. After natural delivery, newborn pups were collected, and genomic DNA was extracted from their ear margin or tail tip tissue. Specific PCR primers targeting the target gene (HRP fusion antibody fragment) were designed, and the target fragment was amplified by PCR. The size of the amplified products was analyzed by agarose gel electrophoresis, and samples with positive amplification bands were selected to confirm whether the target gene was accurately integrated into the rabbit genome, thus obtaining transgenic rabbit individuals carrying the biological fusion enzyme antibody. In short, 68 embryos with the HRP-specific gene sequence were transferred to 4 recipients, 3 of which became pregnant and produced a total of 9 offspring. Among these offspring, 3 were found to have the HRP-specific gene sequence knocked in.

[0121] Table 6. Offspring with homozygous knock-in HRP-specific genes

[0122] Number of transferred embryos Number of receptors Number of pregnancies Number of infants born Enter the number of larvae Male starting number Starting number of females 68 4 3 9 3 1 2

[0123] 1.4 Off-target analysis

[0124] To analyze whether off-target effects exist in HRP protein gene knock-in rabbits, five potential off-target sequences were designed for sgRNA1 based on https: / / crispor.gi.ucsc.edu / , and primers were designed for these five potential off-target sites. The off-target sequence designs are shown in Table 7, and the sgRNA1 off-target site detection primers are shown in Table 8. PCR amplification was used to identify whether off-target effects occurred in HRP gene knock-in rabbits. Sequencing results showed no overlapping peaks near the potential off-target sites, indicating that no off-target effects occurred.

[0125] Table 7

[0126] name Off-target sequences Chromosomal location site Mismatch sgRNA-OT1 GGtGCATTTGCAGCTTTTTCTgTGG chr10 34646056 2 sgRNA-OT2 GGtGCAgGCAGCTCTTTTCTTTTTGG chr12 49830745 2 sgRNA-OT3 aGAGCATGCAGCTTcTTCCTTAGG chr12 91301731 2 sgRNA-OT4 GGAGCAGGTGCAGCTgTTTCTaGGG chr11 54409605 2 sgRNA-OT5 GGAGCcTGGGCAGCTTTcTCTTAGG chr13 38487945 2

[0127] Table 8 Primers for sgRNA1 off-target site detection

[0128]

[0129] 1.5 Performance Validation of Transgenic Rabbits Tagted with Horseradish Peroxidase (HRP) Signaling Protein

[0130] Genotypes of F0 generation rabbits were identified by PCR. Results analysis is shown below. Figure 1 The results showed that F001, F004, and F009 were PCR positive, while the others were negative. The rabbit ear vein serum from F001, F004, F009, and F006 was serially diluted and subjected to TMB substrate color development to verify the results (see...). Figure 2 The results showed that rabbit F006 was negative for PCR and TMB staining, while rabbits F001, F004, and F009 were positive for PCR and TMB staining. The two verification results were consistent.

[0131] 1.6 Genetic stability

[0132] To investigate whether the HRP fusion protein genotype in F0 generation rabbits can be stably inherited by the F1 generation, we crossed F0 generation rabbits with New Zealand White rabbits to obtain F1 generation rabbits. The genotype of the F1 generation was identified by PCR. The results showed that the F0 generation genotype was stably inherited by the F1 generation rabbits. Specific results are as follows: Figure 3 Two rabbits, F108 and F115, were PCR positive.

[0133] Serum from the marginal ear vein of three rabbits (F103, F108, and F115) was collected, and TMB substrate was added for detection. The OD450 readings were as follows. The results showed that the two rabbits (F108 and F115) with positive PCR results had HRP signals, while the rabbit (F103) with negative PCR results did not have HRP signals.

[0134] Table 9. Detection results of HRP signal OD450 in F1 generation rabbits.

[0135] Dilution factor Negative control (F103) F108 F115 200 0.054 1.871 1.846 600 0.034 1.544 1.612 1800 0.063 1.342 1.259 5400 0.049 1.098 1.132 16200 0.062 0.786 0.812 48600 0.046 0.335 0.364 blank 0.052 0.036 0.048

[0136] The HRP gene rabbits with stable genetic information were used for pedigree propagation and population establishment for subsequent experiments.

[0137] Example 2: Preparation of a biofusion enzyme antibody targeting the CD5 target protein

[0138] 2.1 Selection of CD5 protein immunogen sequence

[0139] The CD5 receptor is a 67 kDa type I membrane glycoprotein, structurally belonging to the highly conserved scavenger receptor (SRCR) superfamily. It is expressed in all mature T cells (αβ and γδ) and a small subset of mature B cells (B-1a cells). It is highly expressed in regulatory T cells (Tregs) and regulatory B cells (Bregs) subsets, interacting with T cell and B cell antigen receptors and negatively regulating TCR and BCR-mediated signaling. Based on the CD5 gene sequence published by NCBI, combined with protein structure analysis, two sequences from the extracellular and intracellular regions were selected as immunogens, with the specific design as follows:

[0140] CD5 immunogen regimen one, polypeptide sequence:

[0141] CGGGGNEYSQPPRNSHLSAYPALEGALHRSSMQPDNSSDSDYDLHGAQRL

[0142] CD5 immunogen regimen two, peptide sequence:

[0143] CGGGGGVYLKDGWHMVCSQSWGRSSKQWEDPSQASKVCQRLNCGVPLSLGPFLVTYTPQSSIICYGQLGSFSNCSHSRNDMCHSLGLTCLEPQKTTPPTTRPPPTTT

[0144] The selected polypeptide sequence was sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0145] 2.2 Animal Immunization

[0146] The peptides prepared according to the above methods were conjugated to KLH and BSA vectors, respectively, and immunization was performed according to the following protocol, with specific steps as follows:

[0147] Animal immunization and antiserum detection

[0148] 1) Select 3 rabbits from Example 1 for each group, mark and number the rabbits, collect 300~500ul of blood, and collect the pre-immune serum by centrifugation after coagulation;

[0149] 2) First immunization: Calculate the required antigen volume based on an immunization dose of 300ug per rabbit, emulsify the antigen using an equal volume of Freund's complete adjuvant, and administer immunization via subcutaneous injection at multiple sites;

[0150] 3) Second immunization: Two weeks later, a second immunization is performed. The required antigen volume is calculated based on an immunization dose of 150ug per rabbit. An equal volume of Freund's incomplete adjuvant is used to emulsify the antigen, and multiple subcutaneous injections are used for immunization.

[0151] 4) Third immunization: Two weeks later, the third immunization is carried out. The required antigen volume is calculated based on an immunization dose of 150ug per rabbit. The antigen is emulsified using an equal volume of Freund's incomplete adjuvant and administered via subcutaneous injection at multiple points.

[0152] 5) Serum collection: One week after the third vaccination, 1000~2000ul of blood was collected intravenously, and the serum was collected by centrifugation after coagulation.

[0153] 6) ELISA detection of serum titer: CB was coated with antigen at a final concentration of 1 μg / ml, 100 μl / well, incubated overnight at 4°C, and then blocked with 0.1% casein-PBS. After blocking at room temperature for 1 hour, the plate was washed. Rabbit serum was initially diluted 1:2000 and serially diluted 3-fold. The antibody dilution buffer served as a blank control. Serum titer was detected by ELISA, and the OD450 reading was measured using a microplate reader. The results are shown in Table 10. Single B screening was only performed after the titer reached 1:243000. Ultimately, the titer of the sequence immunization protocol 2 was found to be too low, and rabbit FXCD5101 immunized with the sequence protocol 1 was selected for the next screening step.

[0154] Table 10. CD5 titer ELISA OD values ​​for different immunization regimens

[0155]

[0156] 2.3 The rabbit anti-B screening scheme is detailed in Example 4. After screening, a total of 5 antibodies, CD5-Mab001-005, were finally obtained.

[0157] 2.3.1 Immunohistochemical verification was performed on the cell supernatant from the screening of effective antibodies.

[0158] To further verify the efficacy of the five CD5 cell lines obtained from single B screening in immunohistochemical experiments, different paraffin sections of the same peripheral T-cell lymphoma tissue were selected for preliminary verification using the Dako CD5 antibody (clone number: 4C7), which is widely used in clinical practice. The Dako CD5 antibody procedure was performed according to the manufacturer's instructions: After antigen retrieval using the standard method, incubation with CD5 primary antibody was performed: the optimally diluted primary antibody was added, incubated at 37°C for 60 minutes, and washed three times with 1xPBS for 5 minutes each time; secondary antibody incubation was performed: the optimally diluted secondary antibody was added, incubated at 37°C for 60 minutes, and washed three times with 1xPBS for 5 minutes each time. In this example, the antibody reaction procedure was as follows: incubation with CD5 antibody: the optimally diluted primary antibody was added, incubated at 37°C for 10 minutes, and washed three times with 1xPBS for 0.5 minutes each time. The subsequent staining and counterstaining steps are the same: Add an appropriate amount of freshly prepared DAB staining solution, incubate at room temperature in the dark for 5-10 minutes, observe the staining results under an optical microscope, and do not over-stain. Rinse the sections with tap water to stop the staining process; Counterstaining: Place the sections in hematoxylin for counterstaining, incubate for about 1-5 minutes, wash three times with water, perform rapid destaining treatment with 1% hydrochloric acid ethanol for 30 seconds, and rinse with deionized water for 30 seconds; Dehydration, clearing, and mounting: Immerse the sections sequentially in 75% alcohol for 2 minutes, 85% alcohol for 2 minutes, 95% alcohol for 2 minutes (twice), 100% alcohol for 2 minutes, and xylene for 2 minutes (twice), and mount with neutral resin glue; Microscopic examination and imaging: Image the sections with an optical microscope and take photographs. Results are shown below. Figure 6 .

[0159] pass Figure 6 It can be seen that, under the same working concentration and reaction conditions, the CD5-Mab002 and CD5-Mab005 cell supernatants obtained in the embodiments of the present invention have good affinity and specificity in immunohistochemical experiments. It can be clearly seen that CD5-Mab002 and CD5-Mab005 cells can detect CD5 protein in paraffin sections of peripheral T-cell lymphoma tissue very well. The specificity is comparable to that of the control antibody 4C7, and the staining intensity is better than that of the control antibody 4C7. However, the detection signals of CD5-Mab001, CD5-Mab003, and CD5-Mab004 cells are relatively weak, and the sensitivity for detecting CD5 protein in paraffin sections of peripheral T-cell lymphoma tissue is insufficient.

[0160] 2.3.2 CD5 cell line sequence determination

[0161] The cell supernatants of CD5-Mab002 (clone number PM19A9) and CD5-Mab005 (clone number PM19G6) were sequenced, and the specific procedure is described in Example 4.

[0162] 2.4 Recombinant Antibody Expression

[0163] The CD5-Mab002 (clone number PM19A9) and CD5-Mab 005 (clone number PM19G6) cell lines were sequenced, and the antibodies were recombinantly expressed, as detailed in Example 4. The resulting antibodies were then subjected to TMB substrate staining, and HRP enzyme activity was detected at OD450. It was found that the HRP enzyme activity was higher after antibody expression in the CD5-Mab002 (clone number PM19A9) cell line. The experimental results are shown in Table 11. Finally, CD5-Mab002 was selected for subsequent immunohistochemical experiments to expand and verify the results.

[0164] Table 11 Comparison of HRP activities and OD450 values ​​of two CD5 antibodies

[0165]

[0166] The variable region sequence of the CD5-Mab002 antibody (clone number PM19A9) is as follows:

[0167] CD5-Mab002-VH:

[0168] Nucleotide sequence: SEQ ID NO: 2

[0169] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCAAAAATATGGAATGAGCTGGGTGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGACTGGATAAACACCAACACTGGAGAGCCAACA TATGCTGAAGAGTTCAAGGGACGGTTTGCCTTCTCTTTGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGGAGGGGATGGTGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0170] Amino acid sequence: SEQ ID NO: 1

[0171] QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMSWVKQAPGKGLKWMDWINTNTGEPTYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGGDGAWFAYWGQGTLVTVSA

[0172] CD5-Mab002-VL:

[0173] Nucleotide sequence: SEQ ID NO: 4

[0174] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATGGCTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0175] Amino acid sequence: SEQ ID NO: 3

[0176] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYGYPLTFGAGTKLELK

[0177] 2.5 Post-expression antibody expansion verification

[0178] Paraffin sections tested with three types of CD5 antibodies (clone number: 4C7) from Dako were dewaxed, hydrated, and repaired using standard methods. Blocking buffer (PBS containing 5% goat serum) was added for 1 minute. After rinsing, the working solution of the CD5 fusion enzyme monoclonal antibody carrying the HRP signal protein was directly added. The reaction was carried out at 37°C for 10 minutes. After rinsing, DAB staining solution was added, and staining was carried out at room temperature in the dark for 2-3 minutes. The staining results were observed under an optical microscope; avoid over-staining. Staining was stopped by rinsing with tap water. Hematoxylin was counterstained for 3 minutes, followed by rinsing three times with tap water. Differentiation solution was applied for 15 seconds, followed by rinsing three times with tap water. Ammonia was used for blueing for 30 seconds, followed by rinsing three times with tap water. The sections were then dehydrated, mounted, and photographed. The experimental results are shown below. Figure 7 Experimental results show that using the CD5-Mab002 monoclonal antibody containing the HRP signal protein prepared in this invention for immunohistochemical experiments significantly shortens the reaction time compared to the traditional two-step and three-step immunohistochemical experiments, which take 4-6 hours, without affecting the experimental results. This makes it possible to complete the immunohistochemical experiment on paraffin sections in about 40-60 minutes. Simultaneously, from... Figure 7 As can be seen from the results, the CD5-Mab002 (clone number PM19A9) antibody obtained in this embodiment has comparable sensitivity and specificity to the control antibody.

[0179] Example 3 Preparation of biofusion enzyme antibody targeting TSH target protein

[0180] 3.1 Selection of TSH protein immunogen sequence

[0181] Thyroid-stimulating hormone (TSH) is a glycoprotein secreted by the anterior pituitary gland. It has a molecular weight of approximately 28,000 Daltons and is synthesized by basophilic cells (thyroid-stimulating cells) in the anterior pituitary gland. TSH consists of two non-covalently linked subunits, α and β. The α subunit carries species-specific information and shares similarities with certain amino acid sequences on the α chain of LH, FSH, and hCG. The β subunit carries TSH-specific immunological and biological information. Biological activity requires the simultaneous presence of both α and β subunits.

[0182] TSH testing is a primary screening test for thyroid function. Even small changes in free thyroid hormone concentration can lead to significant adjustments in TSH concentration in the opposite direction. Therefore, TSH is a highly sensitive and specific parameter for testing thyroid function, particularly suitable for early detection or exclusion of dysfunction in the hypothalamic-pituitary-thyroid regulatory loop. TSH measurement is primarily used to: 1) rule out hypothyroidism (elevated TSH levels) or hyperthyroidism (decreased or undetectable TSH levels); 2) monitor T4 replacement therapy in primary hypothyroidism or antithyroid treatment in hyperthyroidism; 3) track the T4 suppression effect of TSH on nutritional factors in "cold nodules" and non-toxic goiter; and 4) assess the response to TRH stimulation tests. With the emergence of more sensitive and accurate testing methods, TSH measurement is increasingly used to identify subclinical or latent hypothyroidism or hyperthyroidism.

[0183] In this embodiment, we selected the full-length human TSH protein sequence published by NCBI, constructed it into the PTT5 vector, and transfected it into the eukaryotic HEK293 cell system for recombinant expression. The experimental procedure was carried out according to the specific method in J. Sambrook's book "Molecular Cloning: A Laboratory Manual" (4th edition).

[0184] 3.2 Animal Immunization

[0185] The recombinant protein prepared according to the above method was then used for immunization according to the following procedure:

[0186] Animal immunization and antiserum detection

[0187] 1) Select 3 rabbits carrying the HRP gene obtained in Example 1, mark and number the rabbits, collect 300~500ul of blood, and collect the pre-immune serum after coagulation and centrifugation;

[0188] 2) First immunization: Calculate the required antigen volume based on an immunization dose of 300ug per rabbit, emulsify the antigen using an equal volume of Freund's complete adjuvant, and administer immunization via subcutaneous injection at multiple sites;

[0189] 3) Second immunization: Two weeks later, a second immunization is performed. The required antigen volume is calculated based on an immunization dose of 150ug per rabbit. An equal volume of Freund's incomplete adjuvant is used to emulsify the antigen, and multiple subcutaneous injections are used for immunization.

[0190] 4) Third immunization: Two weeks later, the third immunization is carried out. The required antigen volume is calculated based on an immunization dose of 150ug per rabbit. The antigen is emulsified using an equal volume of Freund's incomplete adjuvant and administered via subcutaneous injection at multiple points.

[0191] 5) Serum collection: One week after the third vaccination, 1000~2000ul of blood was collected intravenously, and the serum was collected by centrifugation after coagulation.

[0192] 6) ELISA detection of serum titer: CB was coated with antigen at a final concentration of 1 μg / ml, 100 μl / well, incubated overnight at 4°C, and then blocked with 0.1% casein-PBS. After blocking at room temperature for 1 hour, the plate was washed. Rabbit serum was initially diluted 1:2000, serially diluted 3-fold, with antibody dilution as a blank control. Serum titer was detected by ELISA, and OD450 readings were measured using a microplate reader. Single-B screening was performed only after the titer reached 1:243000. Rabbits using the sequence immunization protocol 1 (FXTSH001) were ultimately selected for the next screening step.

[0193] 3.4 The rabbit anti-B screening scheme is detailed in Example 4. In this example, a total of 8 antibodies, TSH-Mab001-008, were finally obtained.

[0194] 3.5 Recombinant Antibody Expression

[0195] The TSH-Mab001-TSH-Mab 008 cell lines were sequenced, and the antibodies were recombinantly expressed, as detailed in Example 4. The resulting antibodies were then subjected to TMB substrate color development, and HRP enzyme activity was detected at OD450. It was found that the HRP enzyme activity was higher after expression of antibodies from TSH-Mab005, TSH-Mab007, and TSH-Mab008 cell lines. The experimental results are shown in Table 12 below.

[0196] Table 12 HRP activity assay results for different TSH antibodies (OD450)

[0197]

[0198] The antibody sequences with the highest enzyme activity, TSH-Mab005, TSH-Mab007, and TSH-Mab008, are as follows:

[0199] The TSH-Mab005 variable region sequence is as follows:

[0200] TSH-Mab005-VH nucleotide sequence: SEQ ID NO: 6

[0201] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATACACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAACCAACATATTCAGATGACTTCAGGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTACAGATCAACAACGTCAAAAATGAGGACACGGGTACATATTTCTGTGGTCAAACTGCGAAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0202] Amino acid sequence of TSH-Mab005-VH: SEQ ID NO: 5

[0203] QIQLVQSGPELKKPGETVKISCKASGYTFTDYSIHWVKQAPGKGLKWMGWINTETGEPTYSDDFRGRFAFSLETSASTAYLQINNVKNEDTGTYFCGQTAKFAYWGQGTLVTVSA

[0204] Nucleotide sequence of TSH-Mab005-VL: SEQ ID NO: 8

[0205] GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAATTCCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGGCGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0206] TSH-Mab005-VL Amino Acid Sequence: SEQ ID NO: 7

[0207] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNSYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPAFGGGTKLEIK

[0208] The variable region sequence of TSH-Mab007 is as follows:

[0209] TSH-Mab007-VH Nucleotide Sequence: SEQ ID NO: 10

[0210] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAGGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAGCTGCTGGAATGCAGTGGGTGCAAAAGATGCCAGGAAAGGGTTTGAAGTGGATTGGCTGGATAAACACCCACTCTGGAGTGCCAAAATATGCAGAAGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCATATTTACAGATAAGCAACCTCAAAAATGAGGACACGGCTACGTATTTCTGTGCGAGTGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0211] TSH-Mab007-VH Amino Acid Sequence: SEQ ID NO: 9

[0212] QIQLVQSGPELKKPGETVRISCKASGYTFTAAGMQWVQKMPGKGLKWIGWINTHSGVPKYAEDFKGRFAFSLETSASTAYLQISNLKNEDTATYFCASGFAYWGQGTLVTVSA

[0213] TSH-Mab007-VL Nucleotide Sequence: SEQ ID NO: 12

[0214] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAAATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0215] Amino acid sequence of TSH-Mab007-VL: SEQ ID NO: 11

[0216] DVVMTQTPLTLSVTIGQPASISCKSSQSLLNSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK

[0217] The variable region sequence of TSH-Mab008 is as follows:

[0218] Nucleotide sequence of TSH-Mab008-VH: SEQ ID NO: 14

[0219] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAACACTTCTAATATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGTTTCACATTATGTGGAATGATAATAAATACTATAATCCAGCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTACAACAACCGGGTATTCCTCAGGATCGCCAATGTGGACACTGCAGATACTGCCACATACTACTGTGCTCGATTCTATAGGTACGCCGGGTATGCTATGGACTCCTGGGGTCACGGAACCTCAGTCACCGTCTCCTCA

[0220] TSH-Mab008-VH amino acid sequence: SEQ ID NO: 13

[0221] QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSNMGVGWIRQPSGKGLEWLFHIMWNDNKYYNPALKSRLTISKDTYNNRVFLRIANVDTADTATYYCARFYRYAGYAMDSWGHGTSVTVSS

[0222] TSH-Mab008-VL nucleotide sequence: SEQ ID NO: 16

[0223] GACATTGTGCTGACTCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGACAGAGGGCCACCATCTCATGCAGGGCCAGCAAGAGTGTCATTACATCTGGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAGCCTATTACTGTCAGCACAGTTGGGAGCTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAG

[0224] TSH-Mab008-VL amino acid sequence: SEQ ID NO: 15

[0225] DIVLTQSPASLAVSLGQRATISCRASKSVITSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAAAYYCQHSWELPTFGSGTKLEIK

[0226] 3.6 Validation of Antibody Performance

[0227] To better verify the application value of TSH antibodies, recombinant TSH protein was diluted to 0 ng / ml, 1 ng / ml, 10 ng / ml, and 100 ng / ml using a chemiluminescence platform. These eight antibodies were then paired with a TSH antibody (catalog number 100023) provided by Medix for double-antibody sandwich assay. The recombinant TSH protein was used as a quality control. Antibodies were used as coating antibodies to coat magnetic beads. TSH-Mab005, TSH-Mab007, and TSH-Mab008, expressing HRP enzyme activity in this embodiment, were directly used as detection antibodies. The dosage was 50 μL of quality control, 50 μL of magnetic beads, and 50 μL of detection antibody, incubated for 10 min, and washed four times. Measurements showed that TSH-Mab005, TSH-Mab007, and TSH-Mab008 antibodies exhibited excellent signal-to-noise discrimination on the chemiluminescence platform (see Tables 13, 14, and 15). A strong linear correlation was also observed with the TSH recombinant protein concentration (see [reference needed]). Figure 9 , Figure 10 , Figure 11 This makes it possible for chemiluminescence platforms to directly use antibodies with enzyme signals, eliminating the need for chemically coupled enzyme labeling.

[0228] Table 13. Chromium emission values ​​and signal-to-noise ratios for TSH-Mab005 antibody detection (0-100 ng / ml)

[0229]

[0230] Table 14. Chromium emission values ​​and signal-to-noise ratios for TSH-Mab007 antibody detection (0-100 ng / ml)

[0231]

[0232] Table 15. Chromium emission values ​​and signal-to-noise ratios for TSH-Mab008 antibody detection (0-100 ng / ml)

[0233]

[0234] Example 4: Single B-cell cloning and sorting

[0235] 4.1 Isolation of rabbit peripheral blood mononuclear cells (PBMCs)

[0236] The transgenic rabbits from Example 1 were used as the blood source. Whole blood containing EDTA was diluted 2-fold with 1x PBS before density centrifugation on mammalian lymphocyte separation medium (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the cell separation medium provided by Sigma, the manufacturer of the PBMC separation medium. The PBMCs were washed twice with 1x PBS before antibody staining.

[0237] 4.2 EL-4B5 medium

[0238] RPMI 1640 (Pan Biotech, Aidenbach, Germany) is supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2mM glutamine, 1% penicillin / streptomycin solution (PAA, Pasching, Austria), 2mM sodium pyruvate, 10mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05mM β-mercaptoethanol (Gibco, Paisley, Scotland).

[0239] 4.3 Consumption of macrophages / monocytes

[0240] Macrophages and monocytes were consumed via nonspecific adhesion using sterile 6-well plates (cell culture grade). Each well was filled with a maximum of 4 ml of culture medium and up to 6 x 10⁶ cells. 6 Peripheral blood mononuclear cells from immunized rabbits were incubated at 37°C and 5% CO2 for 2 hours. The cells in the supernatant were used for the antigen panning step.

[0241] 4.4 Coating of the plate

[0242] Coat sterile 6-well plates of cell culture with 1 μg / ml antigen protein at room temperature or coat sterile streptoacid-coated 6-well plates with 2 μg / ml biotinylated antigen (Microcoat, Bernried, Germany) for 2 hours, or incubate overnight at 4°C. Wash the plates three times in sterile PBS before use.

[0243] 4.5 B cells enrich on antigen proteins

[0244] Inoculate 6-well tissue culture plates coated with antigen protein with up to 6 × 10⁶ cells per 4 ml of culture medium. 6 Cells were collected and incubated at 37°C and 5% CO2 for 1 h to bind. After the enrichment step, non-adherent cells were removed by carefully washing the wells 1-2 times with 1x PBS. The remaining adherent cells were deadhered with trypsin in an incubator at 37°C for 20 min. Trypsin digestion was terminated with EL-4B5 medium. The cells were then washed twice with the medium. The cells were kept on ice until immunofluorescence staining.

[0245] 4.6 Immunofluorescence staining and flow cytometry

[0246] Single-cell sorting was performed using anti-IgG FITC antibody (AbD Serotec, Germany). For surface staining, cells from the consumption and enrichment steps were incubated with anti-IgG FITC antibody in PBS for 30–60 minutes at 4°C in the dark. After centrifugation, the supernatant was aspirated. PBMCs were centrifuged twice and washed with ice-cold PBS. Finally, the PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Propidium iodide at a concentration of 5 μg / ml (BD Pharmingen, San Diego, CA, USA) was added before FACS analysis to distinguish between dead and live cells.

[0247] Single-cell sorting was performed using a flow cytometer (BDFACSDiscover S8) equipped with a computer and FACSDiva software (BD Biosciences, USA).

[0248] 4.7 B cell culture

[0249] Sorted rabbit B cells were cultured in 200 μl / well EL-4B5 medium containing Pansorbin cells (1:100,000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymoma cell supernatant (MicroCoat, Bernried, Germany), and γ-irradiated mouse EL-4B5 thymoma cells (2.5 × 10⁻⁶ cells / well). 4 B cells were incubated in a 37°C incubator for 7 days at 100 cells / well. The supernatant from the B cell culture was removed for screening. The remaining cells were first screened and harvested immediately, and then frozen in 100 μl of RLT buffer (Qiagen, Hilden, Germany) at -80°C.

[0250] 4.8 B-cell sequencing

[0251] The selected usable cells were sequenced, a process carried out by 10x Genomics.

[0252] 4.9 Antibody Recombinant Expression

[0253] The specific steps are as follows:

[0254] (1) Obtaining recombinant antibody genes

[0255] The obtained heavy chain variable region gene VH was used to construct the heavy chain sequence VH-XXFX of recombinant XX antibody (where XX represents the name of the target protein), and the obtained light chain variable region gene VL was used to construct the light chain sequence VL-XXFX of recombinant XX antibody.

[0256] EcoRI and BamHI restriction sites were designed at both ends of the synthesized gene fragment. Using FXRAB prepared by Nanjing Fuxiao Biotechnology Co., Ltd. as a vector, plasmids carrying recombinant genes were obtained as FXRAB-VL (light chain) and FXRAB-VH (heavy chain).

[0257] The heavy chain sequence VH-XXFX and the light chain sequence VL-XXFX were both synthesized by Anhui General Biotechnology Co., Ltd.

[0258] The specific steps are as follows:

[0259] 1) Prokaryotic plasmids containing heavy and light chain sequences were chemically transformed into competent cells and cultured for 14 hours on low-salt LB solid medium containing 100 μg / ml Ampicillin antibiotic. Several single colonies were picked and cultured overnight in low-salt LB liquid medium containing 100 μg / ml Ampicillin antibiotic. Colony PCR was performed using sequencing primers, and 1% agarose gel electrophoresis confirmed that the target gene was successfully inserted into the eukaryotic expression vector.

[0260] 2) The purified plasmid was digested with EcoRI and BamHI. After digestion, 1 μL DpnI was added to the reaction system and the reaction was carried out at 37℃ for 30 min to digest the template DNA.

[0261] 3) The enzyme digestion products were analyzed by 1% agarose gel electrophoresis. The target fragment was excised and then extracted and recovered from the target fragment and the PTT5 vector fragment using a PCR product kit (FastPure GEL DNA DC301-01 Vazyme).

[0262] 4) To carry out a connection reaction;

[0263] The ligation reaction mixture consisted of approximately 0.1 pmol of the target fragment, approximately 0.01 pmol of the FXRAB vector DNA fragment, 1 μL of T4 DNA Ligase buffer (NEB), 1 μL of T4 DNA Ligase (NEB), and deionized water to a final volume of 10 μL. Ligation was performed overnight at 16°C. 3 μL of the reaction product was then transformed into 100 μL of competent DH5α (Vazyme) cells.

[0264] (2) Screening to obtain recombinant plasmids;

[0265] Positive clones were selected, cultured at 37°C for 12 hours, and then plasmids were extracted using a plasmid miniprep kit (Qigen). The extracted plasmids were then sent to Shanghai Sangon Biotech for sequencing and identification.

[0266] (3) Recombinant antibodies against specific target proteins of immunogens were obtained by transfecting CHO cells with recombinant plasmids.

[0267] The correctly sequenced positive clones were cultured in large quantities, and plasmids were extracted according to the (Tiangen DP117) instructions. These plasmids were then co-transfected into healthy CHO cells. 30 μg of the target plasmid was co-transfected into 30 ml of 1×10⁻⁶ cells using the transfection reagent. 6 After expressing antibodies in CHO cells (cells / ml), the culture medium was replaced with fresh medium and cultured for another 5 days. The culture fraction was centrifuged at 4000 rpm and 22°C for 10 minutes, and the culture supernatant was collected. The supernatant was filtered using a 0.45 μM pore size filter tube (Millipore) and then purified using a protein A agarose gel (Changzhou Tiandi Renhe Biotechnology Co., Ltd.). The antibody was analyzed and verified by polyacrylamide gel electrophoresis (SDS-PAGE, 5% stacking gel at 80V, 12% separating gel at 120V) to determine the antibody level and calculate the antibody expression level.

[0268] The antibody was successfully expressed. Under non-reducing conditions, the disulfide bonds were not broken, and the light and heavy chains of the antibody could not be opened. The addition of DTT could open the disulfide bonds to form a linear structure. The expressed antibody was diluted 3 times at a starting concentration of 1:2000. TMB substrate was added to detect HRP activity. The antibody was then identified as having the HRP tag and being successfully expressed based on the TMB colorimetric signal.

[0269] Example 5: Construction of transgenic mice tagged with horseradish peroxidase (HRP) signaling protein using CRISPR / Cas9 technology.

[0270] 5.1 Construction of sgRNA plasmid

[0271] (1) Design the corresponding sgRNA

[0272] Based on the mouse Rosa26 genome sequence, sgRNAs were designed using the online gene editing target site design website CRISPOR (http: / / crispor.tefor.net / ). The design results are as follows:

[0273] sgRNA1: AACGGCTCCACCACGCTCGG (PAM sequence AGG)

[0274] sgRNA2: CCCGATCCCCTACCTAGCCG (PAM sequence AGG)

[0275] sgRNA3: CCTGTCCTCAAGGAATGATC (PAM sequence CGG)

[0276] (2) Design sgRNA amplification primers

[0277] The sgRNA was ligated into the lentiGuide-Puro (Addgene plasmid number 52963) vector to construct the sgRNA-Cas9 expression vector.

[0278] Table 16

[0279]

[0280] After the primer sequences were sent to the company for synthesis, they were annealed and cooled to form double strands, which were then ligated into the linearized plasmid lentiGuide-Puro, which was digested and purified by BsmBI. The ligation status of the vector was then confirmed by PCR and Sanger sequencing.

[0281] 5.2 In vitro cleavage verification of sgRNA cleavage efficiency

[0282] (1) In vitro transcription of sgRNA

[0283] Table 17

[0284]

[0285] Using the sgRNA-Cas9 vector DNA as a template, PCR amplification was performed to obtain an in vitro transcription template for sgRNA. The annealing temperature was 60 °C and the extension time was 5 s. The amplification product was recovered, and in vitro transcription was performed according to the MEGAscript™ T7 transcription kit system and operating procedures. DNA was removed by adding DNase, and RNA was purified using the Vazyme RNA purification kit. The quality of the transcribed RNA was verified by agarose gel electrophoresis.

[0286] (2) Determining cutting efficiency by external cutting

[0287] Using mouse genomic DNA as a template, the target sequence was amplified by PCR. Primer sequences are shown in Table 18. The annealing temperature was 55℃. The amplified product was recovered and used as a substrate to add Cas9 protein (purchased from GenScript USA Inc.) and transcripts for in vitro cleavage. After the cleavage reaction, agarose gel electrophoresis was used to determine the band size, and image analysis of the band grayscale was used to determine the cleavage efficiency. In this example, sgRNA1 showed the highest cleavage efficiency; therefore, subsequent off-target site design was based on predicting off-target sites using sgRNA1.

[0288] Table 18

[0289]

[0290] The specific steps are shown in Table 19.

[0291] Table 19

[0292]

[0293] 5.3 Identify and detect off-target sites

[0294] Potential off-target sites are predicted using the Cas-OFFinder online website (http: / / www.rgenome.net / cas-offinder / ).

[0295] The predicted off-target sites for sgRNA1 are shown in Table 20.

[0296] Table 20

[0297]

[0298] Primers for detecting potential off-target sites of sgRNA1 are shown in Table 21.

[0299] Table 21

[0300]

[0301] 5.4 Constructing the Donor plasmid

[0302] (1) Design homologous arm fragments and synthesize templates.

[0303] The Donor plasmid was selected as FXpcDNA3.1(+). The plasmid was double-digested with ECORⅠ and XhoⅠ restriction endonucleases according to the corresponding system, and then purified.

[0304] Table 22

[0305]

[0306] Based on the restriction enzyme sites, two homologous arms were designed in the mouse genome: the sequence upstream of the insertion site was truncated to create the left homologous arm (HA-L); the sequence downstream of the insertion site was truncated to create the right homologous arm (HA-R). The left and right homologous arms were added to the target fragment, and the template was synthesized by the company (HA-L 574bp HA-R 1127bp for sgRNA1, with the PAM sequence replaced by CGG and the target fragment added in the middle; HA-L 652bp HA-R 1049bp for sgRNA2, with the PAM sequence replaced by CGG and the target fragment added in the middle).

[0307] The amplification primers here are:

[0308] sgRNA1-HA-LF: AATTATGGACTCAACTTGCACGAACAC

[0309] sgRNA1-HA-RR:TCGAGCAGCAGCCATCTGAGATAGGAA

[0310] (2) Carrier connection

[0311] The recovered and purified amplified fragments and linearized FXpcDNA3.1(+) plasmid fragments were ligated using the enzyme ligation system in Table 23. The ligation was then preliminarily verified by transformation, picking of positive clones, and bacterial PCR, and further confirmed by Sanger sequencing.

[0312] Table 23

[0313]

[0314] 5.5 Co-transfection of mouse somatic cells

[0315] The Cas9 / sgRNA plasmid and the Donor plasmid were co-transfected into mouse somatic cells. The transfection efficiency was determined by puromycin selection and fluorescence detection. The target gene was accurately integrated into the mouse genome as verified in Example 1, thereby obtaining transgenic mice carrying the biological fusion enzyme antibody.

[0316] Example 6: Construction of transgenic alpacas with horseradish peroxidase (HRP) signaling protein tags using CRISPR / Cas9 technology

[0317] 6.1 Constructing sgRNA plasmids

[0318] (1) Design the corresponding sgRNA

[0319] Based on the alpaca MSTN genome sequence, sgRNAs were designed using the online gene editing target site design website CRISPOR (http: / / crispor.tefor.net / ). The design results are as follows:

[0320] sgRNA1: TGATGATTACCACGCTACGA (PAM sequence CGG)

[0321] sgRNA2: CAGCGATCTACTACCATAGC (PAM sequence TGG)

[0322] sgRNA3: ACCATGGCCAAGGTATAAGT (PAM sequence AGG)

[0323] (2) Design sgRNA amplification primers

[0324] The sgRNA was ligated into the lentiGuide-Puro (Addgene plasmid number 52963) vector to construct the sgRNA-Cas9 expression vector.

[0325] Table 24

[0326]

[0327] After the above primer sequences were sent to the company for synthesis, they were annealed and cooled to form double strands, which were then ligated into the linearized plasmid lentiGuide-Puro, which was digested and purified by BsmBI. The ligation status of the vector was then confirmed by PCR.

[0328] 6.2 In vitro cleavage verification of sgRNA cleavage efficiency

[0329] (1) In vitro transcription of sgRNA

[0330] Table 25

[0331]

[0332] Using the sgRNA-Cas9 vector DNA as a template, PCR amplification was performed to obtain an in vitro transcription template for sgRNA. The annealing temperature was 60 °C and the extension time was 5 s. The amplification product was recovered, and in vitro transcription was performed according to the MEGAscript™ T7 transcription kit system and operating procedures. DNA was removed by adding DNase, and RNA was purified using the Vazyme RNA purification kit. The quality of the transcribed RNA was verified by agarose gel electrophoresis.

[0333] (2) Determining cutting efficiency by external cutting

[0334] Using alpaca genomic DNA as a template, the target sequence was amplified by PCR. Primer sequences are shown in Table 26. The annealing temperature was 55℃. The amplified product was recovered and used as a substrate to add Cas9 protein (purchased from GenScript USA Inc.) and transcripts for in vitro cleavage. After the cleavage reaction, agarose gel electrophoresis was used to determine the band size, and image analysis of the band grayscale was used to determine the cleavage efficiency. In this example, sgRNA1 showed the highest cleavage efficiency; therefore, subsequent off-target site design was based on predicting off-target sites using sgRNA1.

[0335] Table 26

[0336]

[0337] The specific steps are shown in Table 27.

[0338] Table 27

[0339]

[0340] 6.3 Identify and detect off-target sites

[0341] Based on the prediction of potential off-target sites using the Cas-OFFinder online website (http: / / www.rgenome.net / cas-offinder / ), the predicted off-target sites for sgRNA1 are shown in Table 28.

[0342] Table 28

[0343]

[0344] Primers for detecting potential off-target sites of sgRNA1 are shown in Table 29.

[0345] Table 29

[0346]

[0347] 6.4 Constructing the Donor plasmid

[0348] (1) Synthesize and amplify the target fragment template (including homologous arms)

[0349] The Donor plasmid was selected as FXpcDNA3.1(+). The plasmid was double-digested with ECORⅠ and XhoⅠ restriction endonucleases according to the corresponding system, and then purified.

[0350] Table 30

[0351]

[0352] Based on the restriction enzyme sites, two homologous arms were designed in the alpaca genome: the sequence upstream of the insertion site was truncated to create the left homologous arm (HA-L); the sequence downstream of the insertion site was truncated to create the right homologous arm (HA-R). The left and right homologous arms were added to the target fragment, and the template was synthesized by a company (HA-L 551bp HA-R 863bp for sgRNA1, with the PAM sequence replaced by AGG and the target fragment added in the middle of the right homologous arm). Subsequently, the fragment synthesized by the company was amplified using the following primers.

[0353] sgRNA1 / 3-HA-LF: AATTAGCCAATCATAGATCCTGAC

[0354] sgRNA1 / 3-HA-RR: TCGAAGTAACAGTCCTCCCTTCC

[0355] The sgRNA1 and sgRNA2 genomes are far apart, requiring redesigned left and right homologous arms and amplification primers containing the target fragment. The sgRNA2 fragment synthesized by the company includes (replacing the PAM sequence with AGG on the left homologous arm, sgRNA2 HA-L 601bp HA-R 1215bp, with the target fragment added in the middle).

[0356] sgRNA-HA-LF: AATTAAACTGGGGAAAACAAGAA

[0357] sgRNA1-HA-RR:TCGACATTCACATTATACAGCCATC

[0358] (2) Carrier connection

[0359] The recovered and purified amplified fragments and linearized FXpcDNA3.1(+) plasmid fragments were ligated using the enzyme ligation system in Table 31. The ligation was then preliminarily verified by transformation, picking of positive clones, and bacterial PCR, and further confirmed by Sanger sequencing.

[0360] Table 31

[0361]

[0362] 6.5 Co-transfection of alpaca somatic cells

[0363] The sgRNA plasmid and Donor plasmid were co-transfected into alpaca somatic cells, and the transfection efficiency was determined by puromycin selection and fluorescence detection. The integration of the target gene into the alpaca genome was verified according to Example 1, thereby obtaining transgenic alpaca individuals carrying the biological fusion enzyme antibody.

[0364] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. CD5 antibodies with the signal protein horseradish peroxidase HRP, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding the CD5 antibody with the signal protein horseradish peroxidase (HRP) according to claim 1.

3. The nucleic acid molecule of claim 2, wherein: The nucleotide sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID NO. 2, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 4.

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