Construction method and application of LgBiT transgenic mouse

Abstract

The invention discloses an application of a NanoLuc luciferase double-subunit complementary system in construction of an experimental animal model. The genome of the experimental animal model contains a NanoLuc enzyme large subunit LgBiT encoding gene and stably expresses LgBiT; correspondingly, a tested object used for invading the experimental animal model or connected into the experimental animal model is in fusion connection with NanoLuc luciferase small subunit HiBiT polypeptide serving as a label or a NanoLuc luciferase small subunit HiBiT coding gene serving as a reporter gene, and LgBiT and HiBiT are specifically combined in the experimental animal model and recombined to form the NanoLuc enzyme with catalytic activity. A strong bioluminescence signal is generated under the action of a furazine substrate, so that high-sensitivity, non-invasive and real-time dynamic imaging of in-vivo distribution, loading capacity and diffusion path of a detected object is realized. The invention also discloses a gene editing system for constructing the LgBiT transgenic mouse.

Description

Technical Field

[0001] This invention belongs to the field of medical animal model technology, and relates to the use of the NanoLuc luciferase dual subunit complementary system in constructing experimental animal models, a method for constructing LgBiT transgenic mice, and a gene editing system for constructing LgBiT transgenic mouse models. Background Technology

[0002] In vivo imaging technology enables real-time tracking, visualization, characterization, and measurement of physiological changes in living animals under non-invasive conditions, allowing for qualitative or quantitative studies of biological processes in live animals through imaging methods. Based on its technical principles and applications, it is mainly divided into two categories: functional imaging and structural imaging. Functional imaging includes optical imaging and radio-nuclear imaging; structural imaging encompasses magnetic resonance imaging (MRI), ultrasound imaging, and computed tomography (CT). Compared to structural imaging, which is suitable for anatomical imaging, functional imaging is more suitable for molecular, metabolic, and physiological studies, better reflects the spatial and temporal distribution of cellular or gene expression, and provides a more intuitive understanding of relevant biological processes, specific gene functions, and interactions within living animals.

[0003] Functional in vivo imaging in animals allows for the observation and tracking of target cell, target virus, and gene expression, while simultaneously detecting multiple molecular timeframes. This enables the optimization of drug and gene therapy regimens, observation of drug efficacy at the molecular and cellular levels, assessment of disease and infection progression at the whole animal level, and tracking of the temporal, environmental, developmental, and therapeutic effects on the same animal. Visible light imaging is favored for its non-invasiveness, high sensitivity, high spatiotemporal resolution, low cost, and adaptability. Visible light in vivo imaging technology is primarily based on two revolutionary methods: bioluminescence and fluorescence imaging. Bioluminescence imaging cleverly utilizes luciferase genes to label cells, proteins, and viruses of interest. When luciferase encounters its substrate luciferin, a chemical reaction occurs, producing a bright signal. Fluorescence imaging uses fluorescent proteins or special dyes as markers, which fluoresce under external excitation light. These tissue-penetrating light signals are then captured by a high-precision CCD camera in vitro, processed, and ultimately converted into clear images for researchers to analyze in depth. In contrast, in vivo visible light imaging technology records data from the same group of experimental animals at different time points, tracking dynamic changes within the animal and providing more intuitive and realistic data. Furthermore, this technology does not involve radioactive materials, is simple to operate, and is widely used in life sciences, medical research, and other fields.

[0004] Bioluminescence is a ubiquitous phenomenon in nature, observed in insects, bacteria, fungi, and some marine organisms. This luminescent behavior facilitates a variety of physiological activities, including communication, camouflage, attracting prey, and repelling predators. Since the 1990s, non-invasive imaging has been widely explored for biomedical applications. A bioluminescent system is an enzymatic reaction involving a luciferase and a substrate called luciferin. During this process, with the participation of oxygen, the two-component enzymatic reaction converts chemical energy into light energy, achieving light emission. Compared with traditional fluorescence imaging, bioluminescence has significant advantages: low toxicity, high sensitivity, no need for external light source, and high photon yield. The earliest bioluminescent system used in biomedicine was a species from the beetle *Pyrophorus plagiophthalamus*; however, its large size greatly hindered the study of small molecules or compounds. Subsequently, researchers discovered and developed even smaller luciferases. The smallest luciferase to date, Nanoluciferase (NLuc), was discovered from the deep-sea shrimp *Oplophorus gracilirostris*, with a size of only 19 kDa, making it an important complement to bioluminescent tools. Its small size makes it less invasive to samples when labeling cells, proteins, and viruses, helping to maintain the original sample's natural state. Simultaneously, NLuc exhibits extremely high luminescence intensity and a very bright light signal, making detection in low-concentration samples more sensitive, facilitating the detection of low-expressed target substances and improving analytical sensitivity. Furthermore, compared to other luciferases, NLuc demonstrates significant advantages in rapid reaction, low background luminescence, and versatility.

[0005] NanoBiT is a dual-luciferase complement technology derived from NLuc, consisting of a large subunit (LgBiT, 18 kDa) and a small subunit (HiBiT, 11 amino acids). These two subunits have a strong affinity and can spontaneously complement each other to form a catalytically active NLuc. In the presence of the substrate furazolidone, it produces a bright luminescent signal with intensity proportional to concentration, exhibiting a linear range exceeding seven orders of magnitude, and a luminescence duration exceeding that of existing luciferases. The insertion of only the 11-amino acid HiBiT subunit causes minimal interference with the structure and function of the target substance, further reducing functional alterations caused by tag fusion. Currently, it plays an important role in in vitro studies of protein-protein interactions, protein-ligand interactions, gene regulation and signal transduction, in vitro pathogen infection and drug screening, and tumor killing.

[0006] Bioluminescence imaging is increasingly popular in biomedical research. However, traditional North American firefly-derived Fluc is limited for studying small proteins or compounds due to its high content of hydrophobic amino acids and large molecular weight (61 kDa). Furthermore, FLuc signals decay rapidly, resulting in a narrow imaging window and requiring highly skilled operators. Particularly in imaging viral infection in small animals, many viruses, such as influenza viruses, are highly sensitive to genomic changes, making it impossible to insert large traditional reporter genes. Small-sized NLuc overcomes this obstacle and has been used to trace in vivo transmission in a mouse model of influenza virus. As mice lose weight and infection symptoms worsen, the yield of viruses carrying NLuc tags increases, and the bioluminescence intensity is significantly correlated with this. However, even with intact NLuc, viruses carrying intact NLuc may exhibit reduced viral replication capacity, or even fail to be rescued. The insertion of the small NLuc unit HiBiT has been proven successful in rescuing various viruses (influenza virus, hepatitis B virus, meningococcal virus, enterovirus, etc.) with replication capacity comparable to wild-type viruses. Viruses carrying HiBiT can produce high luminescence signals upon encountering LgBiT and furazolidone substrates. Summary of the Invention

[0007] The inventors previously reported infectious clones of enteroviruses EVA71, CVA16, CVA10, and CVA7 carrying the HiBiT reporter gene and their construction methods in patent document CN116218907B. To expand the application of NLuc from the cellular level to the animal model level, enabling the NLuc luciferase dual subunit complementary system to be applied to laboratory animals and constructed as a visible light imaging technology platform for studying gene regulation mechanisms, drug screening, cell therapy efficacy evaluation, vaccine efficacy verification, and pathogen infection dynamics, we constructed transgenic mice carrying the large subunit LgBiT using C57BL / 6 mice as a background. The target substance carrying HiBiT was injected into these transgenic mice, and the expression and distribution of the target substance could be directly determined using the substrate furazolidone. Compared to the traditional method of euthanizing mice and harvesting tissue to determine gene or virus expression and distribution, this transgenic mouse can rapidly and sensitively capture the distribution and diffusion trajectory of the target substance in the body, and can be continuously observed without affecting the animal's physiological state or experimental progress, reducing the amount of animals used and minimizing experimental harm to mice.

[0008] Therefore, the first objective of this invention is to provide the use of the Nanoluciferase (NanoLuc) dual-subunit complementary system in constructing experimental animal models for studying pathogen invasion, pathogen proliferation or metabolism and death within the body, pathogen transfer and distribution, and the infection and distribution of protein drugs, nucleic acid drugs, and vaccines. These experimental animal models can then be used for pathological research, drug screening, etc., for example, to study the physiological functions of pathogens in vivo, viral replication and distribution in vivo, target tracing and distribution in vivo, evaluation of antiviral and antibacterial efficacy, evaluation of candidate protein drugs and candidate nucleic acid drugs, in vivo distribution and infection / transfection efficiency evaluation of viral / bacterial vector vaccines and DNA / mRNA vaccines, and evaluation of vaccine protective efficiency.

[0009] In one embodiment, the genome of the above-mentioned experimental animal model contains the gene encoding the large subunit LgBiT of the NanoLuc enzyme (nucleotide sequence as shown in SEQ ID NO: 1), and the experimental animal model stably expresses LgBiT (amino acid sequence as shown in SEQ ID NO: 2):

[0010] GTCTTCACACTCGAAGATTTCGTTGGGGACTGGGAACAGACAGCCGCCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGCTGCAGAATCTCGCCGTGTCCGTAACTCCGATCCAAAGGATTGTCCGGAGCGGTGAAAATGCCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGCCGACCAAATGGCCCAGATCGAAGAGGTGTTTAAGGTG GTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCCCTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGCTGAACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCACCCCCGACGGCTCCATGCTGTTCCGAGTAACCATCAACAGCTAA (SEQ ID NO: 1);

[0011] VFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEFFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS (SEQ ID NO: 2).

[0012] Accordingly, the test subjects used to invade or be introduced into experimental animal models are fused with either the NanoLuc luciferase small subunit HiBiT polypeptide (amino acid sequence as shown in SEQ ID NO: 3) as a tag, or the NanoLuc luciferase small subunit HiBiT encoding gene (nucleotide sequence as shown in SEQ ID NO: 4) as a reporter gene.

[0013] VSGWRLFKKIS (SEQ ID NO: 3);

[0014] GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC (SEQ ID NO: 4).

[0015] Optionally, the aforementioned animals include, but are not limited to, clinical medical laboratory animals such as monkeys, pigs, dogs, sheep, rabbits, mice, rats, zebrafish, fruit flies, and insects. For example, the laboratory animal model is a mouse model.

[0016] In one embodiment, the tested object includes pathogens, candidate protein drugs, candidate nucleic acid drugs, or vaccines, selected from the group consisting of: viruses, bacteria, mycoplasma, chlamydia, viral vector vaccines, cell vector vaccines, DNA / mRNA vaccines, pathogenic proteins containing amino acid sequences and / or nucleic acid sequences, candidate protein drugs, candidate DNA or RNA drug molecules, glycoproteins, etc.

[0017] Preferably, the aforementioned viruses include, but are not limited to: adenovirus, poxvirus, adeno-associated virus, hepatitis B virus, influenza virus, Zika virus, tick-borne encephalitis virus, varicella-zoster virus, herpes simplex virus, new Bunyavirus, HIV, monkeypox virus, tick-borne encephalitis virus, yellow fever virus, dengue virus, Venezuelan equine encephalitis virus, Ebola virus, Marburg virus, Rift Valley fever virus, rabies virus, Middle East Respiratory Syndrome Coronavirus, novel coronavirus, severe acute respiratory syndrome coronavirus, Hantavirus, meningococcal B virus, chikungunya virus, poliovirus, norovirus, metapneumovirus, respiratory syncytial virus, mumps virus, parainfluenza virus, rotavirus, Epstein-Barr virus, human papillomavirus, Coxsackie virus, echovirus, enterovirus, human T-cell leukemia virus, lentivirus, etc.

[0018] The aforementioned bacteria include, but are not limited to: Bacillus anthracis, Mycobacterium tuberculosis, Vibrio cholerae, Rickettsia, Yersinia pestis, Acinetobacter baumannii, Bordetella pertussis, Bordetella bronchitidis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira question mark, Mycobacterium leprae, Neisseria gonorrhoeae, Neisseria meningitidis, pathogenic Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Shigella baumannii, etc.

[0019] The chlamydia species are selected from the following group: Chlamydia pneumoniae, Chlamydia trachomatis, etc., but are not limited to this group;

[0020] The mycoplasma mentioned includes, but is not limited to, Mycoplasma pneumoniae.

[0021] The second objective of this invention is to provide a gene editing system for constructing an LgBiT transgenic mouse model, which is a CRISPR / Cas system comprising: a Cas endonuclease, a guide RNA (gRNA) specifically targeting the ROSA26 site of the mouse gene (NCBI accession number NR_027008.1), and a Donor vector containing the LgBiT gene.

[0022] Preferably, in the above gene editing system, the gene sequence of the gRNA is shown in SEQ ID NO: 5:

[0023] CTCCAGTCTTTCTAGAAAGATGGG (SEQ ID NO: 5).

[0024] The LgBiT gene is a polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 1, or a polynucleotide with a nucleotide sequence having ≥80%, preferably ≥85%, preferably ≥90%, preferably ≥95%, more preferably ≥98% homology to SEQ ID NO: 1.

[0025] Optionally, the homologous polynucleotides of the LgBiT gene described above are as shown in any one of SEQ ID NOs: 6-25, including SEQ ID NO: 6 (99% homology), SEQ ID NO: 7 (98% homology), SEQ ID NO: 8 (97% homology), SEQ ID NO: 9 (96% homology), SEQ ID NO: 10 (95% homology), SEQ ID NO: 11 (94% homology), SEQ ID NO: 12 (93% homology), SEQ ID NO: 13 (92% homology), SEQ ID NO: 14 (91% homology), SEQ ID NO: 15 (90% homology), SEQ ID NO: 16 (89% homology), SEQ ID NO: 17 (88% homology), SEQ ID NO: 18 (87% homology), SEQ ID NO: 19 (86% homology), SEQ ID NO: SEQ ID NO: 20 (85% homology), SEQ ID NO: 21 (84% homology), SEQ ID NO: 22 (83% homology), SEQ ID NO: 23 (82% homology), SEQ ID NO: 24 (81% homology), SEQ ID NO: 25 (80% homology).

[0026] The Donor vector is pcDNA3.1-LgBiT, comprising the following fragments sequentially linked: a 2.1 kb 5' homologous arm (SEQ ID NO: 29), a 1.7 kb CAG promoter (SEQ ID NO: 30), a 0.5 kb Kozak-LgBiT (SEQ ID NO: 31), a 0.6 kb WPRE (SEQ ID NO: 32), a 0.3 kb BGH pA (SEQ ID NO: 33), and a 2.2 kb 3' homologous arm (SEQ ID NO: 34).

[0027] Preferably, the above-mentioned endonuclease Cas is Cas 9.

[0028] The aforementioned Cas endonuclease can be in the form of a polypeptide / protein, a polynucleotide containing the Cas encoding gene, or a plasmid pCas used to express the Cas endonuclease; and / or

[0029] The aforementioned gRNAs can be in the form of RNA molecules, polynucleotides containing gRNA-encoding genes, or plasmids used to express gRNAs.

[0030] A third objective of this invention is to provide a kit for constructing an LgBiT transgenic mouse model, comprising the gene editing system described above, and further comprising: a microinjection needle for microinjecting Cas protein, gRNA, and Donor vector into mouse zygotes; and a transplantation tube for transplanting the zygotes into the uterus of a female mouse.

[0031] The aforementioned microinjection needle can be a self-made microinjection needle, such as a microinjection needle tip made by drawing a cored capillary glass tube (SUTTER INSTRUMENT, BF100-78-10) with an outer diameter of 1 mm and an inner diameter of 0.78 mm on a horizontal needle drawing instrument (SUTTER INSTRUMENT, P-97).

[0032] The aforementioned transplant tube can also be a self-made transplant tube, for example, made by calcining a standard melting point measuring glass capillary tube (I-Quip / Core Silicon Valley, C5929-05, inner diameter 0.9-1.1mm, length 120mm).

[0033] Preferably, the above kit may further include a PCR system for verifying LgBiT gene knock-in, the PCR system comprising the following two pairs of primer sequences (1) and (2):

[0034] (1) Forward primer (F1): 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 26),

[0035] Reverse primer (R1): 5'-AGATGTACTGCCAAGTAGGAAAGTC-3' (SEQ ID NO: 27);

[0036] (2) Forward primer (F1): 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 26),

[0037] Reverse primer (R2): 5'-ATACTCCGAGGCGGATCACAA-3' (SEQ ID NO: 28).

[0038] The relationship between PCR amplification products and identification results is as follows:

[0039] Homozygous mice: a 591 bp band (primer pair F1 + R1).

[0040] Heterozygous mice: two bands, 453 bp and 591 bp, respectively.

[0041] Wild-type mice: a 453 bp band (primer pair F1 + R2).

[0042] Furthermore, the above-mentioned kit may also include the following components:

[0043] A viral vector for verifying LgBiT expression in LgBiT transgenic mice, the viral vector containing and expressing the NanoLuc luciferase small subunit HiBiT gene as a reporter gene, wherein the HiBiT gene is a polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 4, or an oligonucleotide with a nucleotide sequence having ≥80%, preferably ≥85%, preferably ≥90%, preferably ≥95%, more preferably ≥98% homology to SEQ ID NO: 4.

[0044] Preferably, the homologous oligonucleotides of the HiBiT gene are as shown in any one of SEQ ID NOs: 35-55, including SEQ ID NO: 35 (97% homology), SEQ ID NO: 36 (97% homology), SEQ ID NO: 37 (97% homology), SEQ ID NO: 38 (94% homology), SEQ ID NO: 39 (94% homology), SEQ ID NO: 40 (94% homology), SEQ ID NO: 41 (92% homology), SEQ ID NO: 42 (92% homology), SEQ ID NO: 43 (92% homology), SEQ ID NO: 44 (89% homology), SEQ ID NO: 45 (89% homology), SEQ ID NO: 46 (89% homology), SEQ ID NO: 47 (86% homology), SEQ ID NO: 48 (86% homology), SEQ ID NO: SEQ ID NO: 49 (86% homology), SEQ ID NO: 50 (83% homology), SEQ ID NO: 51 (83% homology), SEQ ID NO: 52 (83% homology), SEQ ID NO: 53 (81% homology), SEQ ID NO: 54 (81% homology), SEQ ID NO: 55 (81% homology);

[0045] The viral vector is, for example, CMV promoter-KozakHiBiT-GGGGS linker-3×flag with a nucleotide sequence as shown in SEQ ID NO: 56; HBV HiBiT with a nucleotide sequence as shown in SEQ ID NO: 57; CVA10-HiBiT virus reported in patent document CN116218907B; and / or

[0046] NanoLuc luciferase catalyzes the chemiluminescent substrate furazine (FFz).

[0047] Furthermore, in addition to the aforementioned CRISPR / Cas system, biochemical reagents, and experimental equipment, the above-mentioned kit may also include at least one of the following items: a carrying toolbox, the space of which is divided into defined spaces for accommodating one or more containers and operating instruments, such as medicine bottles, test tubes, and the like, each container containing a single component for the method of the present invention; and an instruction manual, which may be written on the bottles, test tubes, and the like, or on a separate piece of paper, or on the outside or inside of the container, such as a paper document with an operation demonstration video APP download window, such as a QR code. The instruction manual may also be in multimedia form, such as a USB flash drive, cloud storage, etc.

[0048] A fourth object of the present invention is to provide a method of using the gene editing system or the kit described above, which is a method for constructing an LgBiT transgenic mouse, comprising the following steps:

[0049] 1) Cas protein, gRNA, and Donor vector were microinjected into mouse zygotes;

[0050] 2) The microinjected fertilized eggs were implanted into the oviducts of surrogate mice for reproduction. After the mice were born, PCR and sequencing were performed to identify LgBiT gene-positive F0 mice.

[0051] 3) Sexually mature positive F0 generation mice were mated with wild-type mice to breed one generation. After the mice were born, they were identified by PCR to obtain LgBiT gene positive F1 generation heterozygous mice.

[0052] 4) Inbreed the F1 generation heterozygous mice and perform PCR identification after the mice are born to obtain LgBiT gene positive F2 generation homozygous mice.

[0053] This invention is the first to apply the NanoLuc luciferase dual-subunit complementary system to an experimental animal model, constructing a transgenic mouse model carrying the NLuc large subunit LgBiT. This solves the problems of ensuring the stability and physiological function of target analytes carrying the HiBiT tag in mice and other animals, as well as their expression, distribution, and diffusion trajectory in vivo. This LgBiT transgenic mouse can provide an efficient and visualized visible light imaging technology platform for gene regulation mechanism analysis, drug screening, cell therapy efficacy evaluation, vaccine efficacy verification, and pathogen infection dynamics research, showing broad application prospects. Attached Figure Description

[0054] Figure 1 A schematic diagram of the target vector construction in the gene editing system used to create LgBiT transgenic mice is shown.

[0055] Figure 2 Electrophoresis diagrams showing genotypic identification of LgBiT transgenic mice are displayed.

[0056] Figure 3 The diagram shows the tracing results of an in vivo bioluminescence verification example in LgBiT transgenic mice carrying the HiBiT expression vector CMV Promoter-Kozak HiBiT-GGGGS linker-3×flag. In the diagram, A: experimental flowchart; B: bioluminescence results in LgBiT transgenic mice.

[0057] Figure 4 The diagram shows the tracing results of the CVA10 HiBiT virus invasion example in LgBiT transgenic mice. A: Experimental flowchart; B: Bioluminescence results of LgBiT transgenic mice.

[0058] Figure 5 The diagram shows the tracing results of an example of HBV HiBiT virus infection in LgBiT transgenic mice. A: Experimental flowchart; B: Bioluminescence results of LgBiT transgenic mice. Detailed Implementation

[0059] This invention applies the NanoLuc luciferase dual-subunit complementary system to experimental animal models. The resulting transgenic mouse model carrying the large NLuc subunit LgBiT can be used as a bioluminescence imaging platform to test viruses, cells, protein molecules, and nucleic acid molecules, overcoming the shortcomings of traditional bioluminescence imaging, such as excessively large reporter gene size, interference with target function, and difficulty in accurately reflecting their life activity characteristics and drug / vaccine efficacy. In vivo gene editing technology is used to knock the large NLuc enzyme subunit LgBiT into the host genome, constructing transgenic animals, such as mouse strains, that stably express LgBiT. As an example of LgBiT transgenic mouse application, when DNA / RNA viruses carrying the HiBiT coding sequence, LNP nanoparticles, exosomes carrying the HiBiT protein / coding sequence, or liposomes are injected into the transgenic mouse, the LgBiT expressed in the mouse specifically binds to HiBiT in vivo, recombining to form the catalytically active NanoLuc enzyme. In the presence of a furazolidone substrate, a strong bioluminescent signal is generated, thereby achieving highly sensitive, non-invasive, real-time dynamic imaging of the in vivo distribution, loading, and diffusion pathway of the target test object. Compared with traditional mouse tissue dissection and in vitro detection, the LgBiT transgenic mouse model constructed in this invention significantly reduces the amount of experimental animals used and avoids interference with the function of the tested virus or target caused by excessively large tracer tags. It provides an efficient and visualized technical platform for gene regulation mechanism analysis, drug screening, cell therapy efficacy evaluation, vaccine efficacy verification, and pathogen infection dynamics research.

[0060] In this document, for the sake of simplicity, the names of proteins such as LgBiT or HiBiT and their encoding genes (DNA) are sometimes used interchangeably. Those skilled in the art should understand that they represent different substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or class of the large subunit of the NLuc enzyme, LgBiT refers to the protein; when used as a gene description, it refers to the gene encoding that large subunit.

[0061] In one specific implementation, an LgBiT transgenic C57BL / 6J mouse model was constructed, and the bioluminescence effect of the mouse model was verified through two pathways: LgBiT gene expression and viral infection. First, LgBiT transgenic mice were constructed by creating LgBiT knock-in mice at the ROSA26 site (NCBI accession number NR_027008.1) of C57BL / 6J mice using CRISPR / Cas-mediated genetic engineering. The mouse ROSA26 gene is located on mouse chromosome 6. The Cas protein, guided by gRNA, binds to the target site, causing a DNA double-strand break. During the repair process, the broken double strands use Donor as a template to generate repair, thereby modifying the target gene. This transgenic mouse can exhibit bioluminescence in the presence of a plasmid carrying HiBiT, a virus, and a furazolidone substrate after injection.

[0062] The process of constructing LgBiT transgenic mice includes the following steps:

[0063] (1) Based on the ROSA26 gene of C57BL / 6J mice, gRNA sequences were designed and Donor vectors containing LgBiT genes were constructed. The gRNA sequences, Donor vectors containing LgBiT genes and Cas proteins were co-injected into fertilized eggs. The microinjected fertilized eggs were returned to the oviducts of surrogate mice for in vivo reproduction. After the mice were born, PCR and sequencing identification were performed to obtain LgBiT gene positive F0 mice.

[0064] (2) The above-mentioned sexually mature positive F0 generation mice were mated with wild-type mice to breed one generation. After the mice were born, PCR identification was performed to obtain LgBiT gene positive F1 generation heterozygous mice.

[0065] (3) The above-mentioned sexually mature positive F0 generation mice were mated with wild-type mice to breed one generation. After the mice were born, PCR identification was performed to obtain LgBiT gene positive F1 generation heterozygous mice.

[0066] When validating the LgBiT knock-in mouse model constructed above, viruses carrying the HiBiT gene can be used, including HiBiT enterovirus and HiBiT hepatitis B virus.

[0067] Compared with the prior art, the present invention has the following advantages:

[0068] 1. The constructed LgBiT transgenic mouse model can express LgBiT protein, which can be used to study target substances carrying HiBiT, including but not limited to target gene and protein function, drug and vaccine screening and evaluation, cell therapy evaluation, and viral infection tracking. It helps to visualize target substances in mice and other animals, achieve sensitive, efficient and rapid detection, and promote the progress of related research.

[0069] 2. Low-dose / subclinical infection tracing. Experiments show that this model can sensitively capture the dynamic processes of viral replication sites, intensity, distribution, and spread of non-susceptible viruses such as CVA10 under conditions of no obvious clinical symptoms or low-dose infection.

[0070] 3. Non-invasive real-time activity monitoring. Experiments show that this model can non-invasively and dynamically reflect the HBV replication activity in liver tissue, overcoming the limitations of traditional methods that cannot detect HBV in situ in the liver.

[0071] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. It should be understood that after reading the concept of the present invention, any changes or adjustments made by those skilled in the art should fall within the protection scope of the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0072] Example

[0073] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0074] Materials and methods

[0075] The primer synthesis and gene sequencing in the examples were performed by Sangon Biotech (Shanghai) Co., Ltd.

[0076] The Cas enzyme was purchased from New England Biolabs (NEB), catalog number: M0668T;

[0077] The gRNA was synthesized by Genscript Biotech Inc.

[0078] C57BL / 6J mice were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd., aged 3-4 weeks and weighing 12-15g.

[0079] Microinjection needle raw material: capillary glass tube with core (SUTTERINSTRUMENT, BF100-78-10) with an outer diameter of 1mm and an inner diameter of 0.78mm.

[0080] Horizontal needle puller (P-97);

[0081] Transplant tube raw material: standard melting point measuring glass capillary tube (I-Quip / Core Silicon Valley, C5929-05, inner diameter 0.9-1.1mm, length 120mm);

[0082] The virus CVA10 HiBiT used in this embodiment is the CVA10-HiBiT reported by the inventor in the patent document with publication number CN116218907B. Part of the contents of that patent document can be incorporated into this document for reference.

[0083] Animal in vivo imaging system: Revvity, Inc., IVIS Lumina Series III.

[0084] Statistical analysis: In this study, all numerical variables are expressed as mean ± standard error. Two-tailed Student's t-tests were used for comparisons between two groups, and ANOVA tests were used for comparisons among three groups. A p-value < 0.05 was considered statistically significant.

[0085] Example 1: Construction of LgBiT transgenic mice

[0086] Based on the mouse ROSA26 gene (NCBI accession number NR_027008.1), gRNA was designed, and a Donor vector containing the LgBiT gene was constructed. The gRNA, the Donor vector containing the LgBiT gene, and the Cas protein were simultaneously microinjected into recipient mouse zygotes. The recipient mouse zygotes were then transplanted into surrogate mice for reproduction. After the mice were born, PCR and sequencing were performed to identify LgBiT gene-positive F0 generation mice.

[0087] The designed gRNA nucleotide sequence is: CTCCAGTCTTTCTA GAAGATGGG (SEQ ID NO: 5).

[0088] The donor vector is pcDNA3.1-LgBiT (this fragment mainly includes a 2.1 kb 5' homologous arm SEQ ID NO: 29, a 1.7 kb CAG promoter SEQ ID NO: 30, a 0.5 kb Kozak-LgBiT SEQ ID NO: 31, a 0.6 kb WPRE SEQ ID NO: 32, a 0.3 kb BGH pA SEQ ID NO: 33, and a 2.2 kb 3' homologous arm SEQ ID NO: 34, as shown in the diagram). Figure 1 As shown.

[0089] Three- to four-week-old female C57BL / 6J mice were injected with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) at an interval of 46 to 48 hours. Following HCG injection, the female mice were mated with adult fertile male mice to achieve fertilization. The following day, the female mice were euthanized, and fertilized eggs were collected from the oviducts and placed in a 37°C, 5% CO2 incubator for later use.

[0090] Prepare a microinjection needle and fixation needle. Add the prepared Donor vector, gRNA, and Cas protein into the microinjection needle. Select morphologically normal fertilized eggs and place them in an injection dish. Under an inverted microscope, inject the Donor vector, gRNA, and Cas protein into the nucleus of the fertilized egg cells. After injection, transfer the fertilized eggs to M16 medium and incubate them at 37°C with 5% CO2 for 0.5–1 h before transplantation.

[0091] To prepare pseudopregnant female mice, select age-appropriate fertile female mice and mate them with vasectomized male mice. Stimulate the female mice to induce a series of pregnancy changes, resulting in pseudopregnant female mice, which will then serve as surrogate mice for transgenic fertilized eggs. The fertilized eggs injected with the exogenous gene are then transferred into the oviduct of the surrogate female mouse on the day of ovulation. Successfully implanted female mice typically give birth 19-21 days after surgery. One week after birth, the sucklings are numbered by clipping their toes and simultaneously identified by PCR.

[0092] Example 2: Identification and passage of LgBiT transgenic mice

[0093] The pups born to surrogate mothers were designated as F0 generation. The toes of 1-week-old pups were collected, and DNA was extracted from the mouse tissues using the TINGEN (DP304) kit. For details of the method, please refer to the instructions in the kit.

[0094] The PCR identification primers consist of two pairs (1) and (2):

[0095] (1) Forward primer (F1): 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 26),

[0096] Reverse primer (R1): 5'-AGATGTACTGCCAAGTAGGAAAGTC-3' (SEQ ID NO: 27).

[0097] (2) Forward primer (F1): 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 26),

[0098] Reverse primer (R2): 5'-ATACTCCGAGGCGGATCACAA-3' (SEQ ID NO: 26).

[0099] The relationship between PCR amplification products and identification results is as follows:

[0100] Homozygous mice: a 591 bp band (primer pair F1 + R1).

[0101] Heterozygous mice: two bands, 453 bp and 591 bp, respectively.

[0102] Wild-type mice: a 453 bp band (primer pair F1 + R2). (e.g.) Figure 2 As shown.

[0103] PCR system components: PCR amplification was performed using the 2×Hieff Ultra-Rapid II HotsStart PCR MasterMix kit (YESEN 10167ES08). The total reaction volume was 50 μl. Specific components are shown in Table 1.

[0104] Table 1. PCR reaction system for mouse genotype identification

[0105] Components volume 2×Hieff Ultra-Rapid II HotsStart PCR Master Mix 25 μl forward primer 2 μl reverse primer 2 μl DNA template 1 μl <![CDATA[ddH2O]]> 20 μl

[0106] The PCR cycle amplification program is shown in Table 2:

[0107] Table 2. PCR amplification procedure for mouse genotype identification

[0108]

[0109] Agarose gel electrophoresis was performed using 50×TAE Buffer (YESEN 60116ES76), prepared as 1×TAE Buffer using ddH2O, and GoldBand DL5,000 DNA Marker (YESEN 10504ES60).

[0110] Based on the above identification results, LgBiT positive homozygous F0 generation mice were obtained, and they were passaged and established.

[0111] Furthermore, F0 generation mice carrying LgBiT were mated with untransgenic mice and passaged to obtain F1 generation mice. After one week of age, toes were harvested and the PCR identification method described above was repeated to obtain LgBiT transgenic heterozygous F1 generation mice. These mice can be used for experiments and further passage.

[0112] Furthermore, homozygous LgBiT transgenic F2 generation mice were obtained. The heterozygous LgBiT transgenic F1 generation mice were mated with their siblings to produce F2 generation mice. After one week of age, the toes of these mice were harvested, and the PCR identification method described above was repeated to obtain homozygous LgBiT transgenic F2 generation mice. These mice can be used for experiments and further passage.

[0113] Furthermore, LgBiT transgenic homozygous F2 generation mice were crossbred with their siblings to obtain 100% LgBiT transgenic homozygous mice, ultimately obtaining the transgenic C57BL / 6J mouse strain with stable LgBiT expression.

[0114] Example 3: Results of in vivo imaging and bioluminescence experiments in mice carrying the HiBiT gene

[0115] Plasmid construction: CMV promoter-Kozak HiBiT-GGGGS linker-3×flag (SEQ ID NO: 56)

[0116] Using the principles of homologous recombination and vector self-ligation, the Kozak HiBiT-GGGGS linker-3×flag gene was directly inserted into a vector containing a CMV promoter.

[0117] Forward primer (F2): 5'-TGGCGGCTGTTCAAGAAGATTAGCGGCGGCGGAGGATCTGACTACAAAGACCATGACG-3',

[0118] Reverse primer (R3): 5'-CTAATCTTCTTGAACAGCCGCCAGCCGCTCACCATGGTGGCGGATCCGGTGGCTCTTAT -3'.

[0119] To simplify the experimental setup, this embodiment uses a tail vein hydrodynamic method to inject the aforementioned vector into LgBiT transgenic mice. Specifically, 20 μg of plasmid was diluted with PBS to 10% of the LgBiT transgenic mouse's body weight and injected via the tail vein. The injection time was maintained at 5-8 seconds to avoid rapid injection that could lead to a rapid increase in peripheral blood volume and subsequent heart failure. In vivo imaging (model: IVIS Lumina Series III) was performed 48 hours after plasmid injection.

[0120] Preparation of imaging substrate: Nano-Glo® Fluorofurimazine In Vivo Substrate (Promega N4100) was used as the imaging substrate. Each vial of FFz (furazine) lyophilized powder was dissolved in 525 μl of PBS and gently mixed with a pipette.

[0121] Mice injected with the plasmid for 48 h were shaved to prevent absorption of luminescence by their fur. The shaved mice were then intraperitoneally injected with 50 μl of the substrate FFz. After injection, the mice were allowed to move for 5 min to allow FFz to circulate and diffuse sufficiently within their bodies. Five min later, the mice were placed in a gas anesthesia container and anesthetized with isoflurane. Once deeply anesthetized, the mice were placed in a small animal in vivo imaging instrument for bioluminescence imaging.

[0122] To demonstrate that the HiBiT-carrying protein can be expressed at the intended site in mice and can bind to LgBiT produced in LgBiT transgenic mice, spontaneously generating photons in the presence of substrate FFz and being captured by a high-precision small animal in vivo imaging system, this example constructed a protein vector expressing HiBiT. The expression vector was injected hydrodynamically via the tail vein to temporarily increase the peripheral blood circulation volume of LgBiT transgenic mice, making it easier for the plasmid to enter the highly permeable liver tissue and express within it for a short period. The injected LgBiT transgenic mice were imaged on days 2 and 4 to observe their bioluminescence.

[0123] Experiments such as Figure 3 As shown, compared to control LgBiT transgenic mice, LgBiT transgenic mice injected with the HiBiT protein expression vector CMV promoter-Kozak HiBiT-GGGGS linker-3×flag exhibited significant bioluminescence on days 2 and 4 post-injection in the presence of substrate FFz, with expression distribution clearly concentrated in liver tissue. For short-term protein expression, the bioluminescence intensity of LgBiT transgenic mice was higher on day 2 than on day 4, indicating that the expression level of the HiBiT-carrying protein expression vector was reduced.

[0124] These results demonstrate that the HiBiT-carrying gene can be expressed in LgBiT transgenic mice, and the expression intensity and distribution of the related gene can be tracked in the presence of the substrate FFz. This further suggests that the distribution and intensity of HiBiT-carrying proteins, small molecule and peptide drugs, and vaccines can all be tracked in LgBiT transgenic mice, providing a sensitive and intuitive animal model for studying the physiological functions of proteins, the pharmacological effects of small molecule and peptide drugs, and the expression distribution and efficacy and safety evaluation of vaccines.

[0125] Example 4: In vivo infection tracking experiment of Coxsackie A10 virus carrying HiBiT in mice

[0126] The Coxsackie A10 virus carrying HiBiT in this embodiment (abbreviated as CVA10 HiBiT) is the CVA10-HiBiT reported by the inventor in the patent document with publication number CN116218907B.

[0127] Female and male LgBiT transgenic mice were housed together to induce pregnancy. Once the female mice showed obvious signs of pregnancy, they were separated from the male mice. After the female mice gave birth, the surrounding environment was kept quiet. On the 7th day after birth (7-day-old mice), they were given 3 × 10⁻⁶ pups. 5 TCID 5050 μl of CVA10 HiBiT was injected intraperitoneally into 7-day-old suckling mice. The 7-day-old suckling mice injected with CVA10 HiBiT virus were then returned to female mice for growth. Small animal in vivo imaging was performed on day 5 after CVA10 HiBiT virus injection.

[0128] Preparation of imaging substrate: Nano-Glo® Fluorofurimazine In Vivo Substrate (Promega N4100) was used as the imaging substrate. Each vial of FFz lyophilized powder was dissolved in 525 μl of PBS and gently mixed with a pipette. The amount of imaging substrate used was adjusted according to the weight of 12-day-old suckling mice and the recommended injection dosage in the imaging substrate instructions.

[0129] Mice were intraperitoneally injected with 25 μl of the substrate FFz. After injection, the mice were allowed to move for 5 minutes to allow FFz to circulate and diffuse sufficiently within their bodies. Five minutes later, the mice were placed in a gas anesthesia container and anesthetized with isoflurane. Once deeply anesthetized, the mice were placed in a small animal in vivo imaging instrument for bioluminescence imaging.

[0130] The results of the visualization experiment on the invasion and infection of CVA10 HiBiT in LgBiT transgenic mice are analyzed as follows:

[0131] Mice are not natural hosts for many human viruses, resulting in weak infectivity or even inability to establish effective infection in mice. Human viruses with weak infectivity and replication in mice often do not produce obvious clinical symptoms in healthy adult mice, only exhibiting transient viral replication. Researchers often require high viral doses and a large number of mice to determine the window period, dosage, and distribution of viral infection. This process necessitates euthanizing and sampling multiple mice at the same time point to determine titers and pathological conditions in various tissues, consuming significant human and material resources. Furthermore, the large number of mice used does not comply with ethical requirements for reducing animal usage. Simultaneously, each mouse only indicates the viral infection and disease progression at a single time point, lacking a complete study of the viral infection and disease progression of an individual mouse. This results in a lack of a comprehensive research system covering the entire process from viral infection to spread, disease development, and even viral clearance and symptom reduction after subsequent treatment. To address this issue, this embodiment involves infecting LgBiT transgenic mice with a virus carrying HiBiT and then observing whether the LgBiT transgenic mice produce bioluminescence under asymptomatic infection conditions, thereby capturing the infection and spread of the virus within the mice.

[0132] This embodiment uses the CVA10-HiBiT virus reported by the inventors in patent document CN116218907B. Specifically, the prototype strain of the CVA10 HiBiT virus belongs to Coxsackievirus A10 (CVA10) of the enterovirus A group. This prototype strain can cause symptoms such as anorexia, low-grade fever, and ulcers on the hands, feet, and mouth in children under 5 years old, i.e., hand-foot-mouth disease. Currently, hand-foot-mouth disease is a Class C infectious disease in China. Although most children recover spontaneously in about one week, a small number of children are at risk of developing complications of the central nervous system, respiratory system, and cardiovascular system, which can lead to death. However, there are currently no specific drugs or preventive vaccines for CVA10 on the market. One important reason is that adult mice are not sensitive to CVA10, and the virus cannot establish effective replication and infection in mice. Currently, most animal model studies on CVA10 focus on suckling mice under one week old, which can induce obvious clinical symptoms and even death in suckling mice. However, this model also faces the aforementioned problems: a high viral infection dose, a large number of suckling mice, and a lack of dynamic data on viral infection transmission and disease development. Furthermore, this patent document describes the construction of a CVA10 infection plasmid carrying HiBiT, successfully rescuing the CVA10 HiBiT virus. This virus can be stably passaged and emits light, demonstrating application potential at the cellular level.

[0133] Therefore, this embodiment uses CVA10 HiBiT 3 × 10 5 TCID 50 Infection was established by intraperitoneal injection into 1-week-old LgBiT transgenic mice. Bioluminescence in the LgBiT transgenic mice was then observed on days 5 and 7 post-infection.

[0134] Experimental results ( Figure 4 The results showed that LgBiT transgenic mice infected with CVA10 HiBiT exhibited no obvious clinical symptoms throughout the observation period, consistent with the experimental objective of capturing short-term infection distribution in the absence of clinical symptoms. The results also indicated that LgBiT transgenic mice infected with CVA10 HiBiT produced bioluminescence on days 5 and 7 post-infection, with the overall bioluminescence intensity on day 5 being stronger than on day 7. However, the viral distribution on day 7 was significantly wider than on day 5. These results suggest that viral replication on day 5 was primarily active in the abdomen, such as the liver and gastrointestinal tract, with a small amount distributed in the lungs. On day 7, the virus was active throughout the body, and compared to day 5, it was more widely distributed in the thigh muscles, limbs, thymus, and lungs. Therefore, it can be inferred that the CVA10 HiBiT virus actively replicated in the abdominal tissues of LgBiT transgenic mice for a short period before spreading throughout the body. During this period, the virus was gradually cleared by the mouse's immune system.

[0135] The above results indicate that after LgBiT transgenic mice are infected with the virus carrying HiBiT, asymptomatic infection can be established in the LgBiT transgenic mice. At the same time, in the presence of the substrate FFz, its bioluminescence can be captured by a small animal in vivo imaging instrument, which can determine the replication site, intensity, and distribution of the virus in the transgenic mice, as well as the dynamic detection process of viral infection.

[0136] Further, this LgBiT transgenic mouse model demonstrates its ability to sensitively capture viral infections that rarely present clinical symptoms, allowing for direct observation of viral distribution and replication intensity. Considering that in the real world, viral invasion of the body often occurs at low doses, typically replicating extensively at the site of invasion or in specific tissues before being released and causing symptoms in other areas, this model can simulate low-dose viral infection under natural conditions, providing researchers with a sensitive animal model for studying real viral invasion and disease development. Furthermore, this LgBiT transgenic mouse can be crossbred with other transgenic mice as needed by researchers to achieve broader applications.

[0137] Example 5: In vivo infection tracking experiment of hepatitis B virus carrying HiBiT (HBV HiBiT) in mice

[0138] The nucleotide sequence of the hepatitis B virus carrying HiBiT (abbreviated as: HBV HiBiT) used in this embodiment is shown in SEQ ID NO: 57.

[0139] Six- to eight-week-old LgBiT mice were injected with HBVHiBiT virus, which was delivered via adeno-associated virus type 8, through the orbital venous plexus. At week 5, in vivo imaging was performed using a small animal imaging system.

[0140] Preparation of imaging substrate: Nano-Glo® Fluorofurimazine In Vivo Substrate (Promega N4100) was used as the imaging substrate. Each vial of FFz lyophilized powder was dissolved in 525 μl of PBS and gently mixed with a pipette. The amount of imaging substrate used should be according to the injection volume recommended in the manufacturer's instructions.

[0141] Mice were injected intraperitoneally with the substrate FFz and then allowed to move for 5 minutes to allow FFz to circulate and diffuse fully within the mice. After 5 minutes, the mice were placed in a gas anesthesia container and anesthetized with isoflurane. Once the mice were deeply anesthetized, they were placed in a small animal in vivo imaging instrument for bioluminescence imaging.

[0142] The results of the visualization experiment on HBV HiBIT infection in LgBiT transgenic mice are analyzed as follows:

[0143] Hepatitis B virus (HBV) is an enveloped, hepatotropic DNA virus with species specificity, naturally infecting only primates. According to the WHO, approximately 2 billion people worldwide have a history of or current HBV infection, with about 240 million being chronically infected. China accounts for about one-third of these infections globally. Animal models are crucial for understanding HBV replication mechanisms and the resulting hepatocellular damage, liver fibrosis, and drug efficacy evaluation. However, traditional HBV infection mouse models can only dynamically detect HBV-related markers (HBsAg, HBeAg, HBV DNA) in mouse blood, failing to directly reflect viral replication and disease progression in liver tissue. This requires sampling from multiple mice at the same time point, lacking a complete picture of the individual's viral infection and disease progression. Furthermore, the persistent presence of covalently closed circular DNA (cccDNA) is a major cause of chronic HBV infection, leading to liver fibrosis and liver cancer. The disappearance of HBV DNA in the blood does not necessarily mean that cccDNA has been cleared from the liver tissue; it may be in a state of transcriptional silencing, making relapse likely after drug discontinuation. Similarly, the disappearance of HBsAg in "functional cure" only means that cccDNA transcriptional activity is nearly silenced. Currently, cccDNA detection is mainly based on liver biopsy. In addition to being invasive, cccDNA is unevenly distributed in the liver, making it difficult to accurately detect the actual activity of cccDNA in liver tissue.

[0144] To accurately reflect HBV replication activity in mice, this embodiment inserts HiBiT into the S protein coding region of the HBV genome and inserts this infectious genome into adeno-associated virus type 8 (AAV8) as a delivery vector, injecting 10 via the orbital venous plexus. 11 Infection of LgBiT transgenic mice was established using GC-dose viral fluid, and bioluminescence in LgBiT transgenic mice was observed at week 5.

[0145] Experimental results show that ( Figure 5 In LgBiT transgenic mice infected with HBV HiBiT, bioluminescence was observed in the liver tissue at week 5, with the signal intensity concentrated in the liver tissue. This indicates that HBV HiBiT can produce bioluminescence in the liver tissue of LgBiT transgenic mice, and this bioluminescence can reflect the active capacity for HBV replication in the liver tissue. These LgBiT transgenic mice can provide a direct indicator for evaluating the "complete cure" of HBV using relevant drugs and cell therapies.

[0146] In summary, the LgBiT transgenic mouse model provided by this invention can effectively bind to protein expression vectors carrying HiBiT and HiBiT expressed or replicated in viruses to form an active luciferase, which spontaneously generates bioluminescence in the presence of the substrate FFz. This provides an intuitive and sensitive dynamic visualization mouse model for subsequent studies on gene expression, protein physiological function, small molecule and peptide drugs, vaccine evaluation, and cell therapy evaluation.

Claims

1. The use of Nanoluciferase, or NanoLuc luciferase dual subunit complementary system, in the construction of experimental animal models, which are used to study pathogen invasion of the body, pathogen proliferation or metabolism and death in the body, pathogen transfer and distribution in the body, and infection and distribution of protein drugs, nucleic acid drugs and vaccines in the body.

2. The use as described in claim 1, characterized in that, The genome of the experimental animal model contains the gene encoding the large subunit LgBiT of the NanoLuc enzyme (nucleotide sequence as shown in SEQ ID NO: 1), and the experimental animal model stably expresses LgBiT (amino acid sequence as shown in SEQ ID NO: 2). Accordingly, the test subject used to invade or be introduced into the experimental animal model is fused with either the NanoLuc luciferase small subunit HiBiT polypeptide (amino acid sequence as shown in SEQ ID NO: 3) as a tag, or the NanoLuc luciferase small subunit HiBiT encoding gene (nucleotide sequence as shown in SEQ ID NO: 4) as a reporter gene.

3. The use as described in claim 1, characterized in that, The animals are selected from monkeys, pigs, dogs, sheep, rabbits, mice, rats, zebrafish, fruit flies, and insects.

4. The use as described in claim 2, characterized in that, The tested objects include pathogens, candidate protein drugs, candidate nucleic acid drugs, or vaccines, selected from the group consisting of: viruses, bacteria, mycoplasma, chlamydia, viral vector vaccines, cell vector vaccines, DNA / mRNA vaccines, pathogenic proteins containing amino acid sequences and / or nucleic acid sequences, candidate protein drugs, candidate DNA or RNA drug molecules, and glycoproteins.

5. A gene editing system for constructing LgBiT transgenic mice, which is a CRISPR / Cas system, comprising: The nuclease Cas, a guide RNA (gRNA) specifically targeting the mouse gene ROSA26 site (NCBI accession number NR_027008.1), and a Donor vector containing the LgBiT gene, wherein the gene sequence of the gRNA is shown in SEQ ID NO:

5.

6. The gene editing system as described in claim 5, characterized in that, The LgBiT gene is a polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 1, or a polynucleotide with a nucleotide sequence having ≥80% homology to SEQ ID NO:

1.

7. The gene editing system as described in claim 6, characterized in that, The Donor vector is pcDNA3.1-LgBiT, comprising the following fragments sequentially linked: a 2.1 kb 5' homologous arm (SEQ ID NO: 29), a 1.7 kb CAG promoter (SEQ ID NO: 30), a 0.5 kb Kozak-LgBiT (SEQ ID NO: 31), a 0.6 kb WPRE (SEQ ID NO: 32), a 0.3 kb BGH pA (SEQ ID NO: 33), and a 2.2 kb 3' homologous arm (SEQ ID NO: 34).

8. A kit for constructing LgBiT transgenic mice, characterized in that, The gene editing system comprising any one of claims 5-7 further comprises: a microinjection needle for microinjecting Cas protein, gRNA and Donor vector into mouse zygotes; and a transplantation tube for transplanting the zygotes into the uterus of a female mouse.

9. The reagent kit as described in claim 8, characterized in that, It also includes a PCR system for verifying the LgBiT gene knock-in, the PCR system comprising the following two pairs of primer sequences (1) and (2): (1) Forward primer (F1): 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 26), Reverse primer (R1): 5'-AGATGTACTGCCAAGTAGGAAAGTC-3' (SEQ ID NO: 27); (2) Forward primer (F1): 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 26), Reverse primer (R2): 5'-ATACTCCGAGGCGGATCACAA-3' (SEQ ID NO: 28).

10. The method of using the gene editing system as described in any one of claims 5-7 or the kit as described in any one of claims 8-9, wherein it is a method for constructing an LgBiT transgenic mouse, characterized in that, Includes the following steps: 1) Cas protein, gRNA, and Donor vector were microinjected into mouse zygotes; 2) The microinjected fertilized eggs were implanted into the oviducts of surrogate mice for reproduction. After the mice were born, PCR and sequencing were performed to identify LgBiT gene-positive F0 mice. 3) Sexually mature positive F0 generation mice were mated with wild-type mice to breed one generation. After the mice were born, they were identified by PCR to obtain LgBiT gene positive F1 generation heterozygous mice. 4) Inbreed the F1 generation heterozygous mice and perform PCR identification after the mice are born to obtain LgBiT gene positive F2 generation homozygous mice.

Images