Modeling method for rapidly preparing humanized monkey model

Abstract

The invention relates to the technical field of biology, in particular to a modeling method for rapidly preparing a humanized monkey model, which comprises the following steps: S1, constructing an AAV9-ACE2-GFP vector, observing the expression condition of GFP, and detecting the expression condition of ACE2 in infected cells in combination with flow cytometry; s2, directionally infecting the lung of the mouse with an AAV9-ACE2 virus, and detecting the RNA expression level of human ACE2 to verify the expression effect of the AAV9-ACE2 virus in the mouse body; s3, the AAV-ACE2 virus is targeted to the lung organ of the monkey, and the expression quantity of the human ACE2 gene in the lung tissue is detected through Q-PCR; and S4, carrying out a novel coronavirus infection experiment by using the humanized monkey model obtained in the step S3. The method comprises the following steps: firstly, establishing a virus delivery mode to carry out humanization on lungs of non-human primates; the genetically modified monkey is further subjected to a challenge experiment, a monkey model more susceptible to novel coronavirus is prepared, and a humanized monkey can be rapidly generated to be used for evaluating novel coronavirus related drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for rapidly preparing a humanized monkey model. Background Art

[0002] Coronaviruses are single-stranded positive-strand RNA viruses with multiple strains, including SARS-CoV-1 (SARS-CoV) and SARS-CoV-2, both of which can cause severe acute respiratory syndrome. SARS-CoV-2 infection of host cells involves the specific binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor of the host cell, so the expression of ACE2 in host cells is the key to viral infection. Suitable animal models are crucial for studying the infection mechanism of SARS-CoV-2 and are also key to evaluating the effectiveness of drugs and vaccines. Bioinformatics analysis found that animals with similar homology to human ACE2 receptors include rhesus monkeys, crab-eating macaques, African green monkeys, hamsters, minks, and cats. Inbred mice are widely used to study gene function or disease mechanisms because of their homogeneity in genetic background and consistency in experimental results. However, due to the differences between mouse and human ACE2, traditional mouse models cannot effectively simulate SARS-CoV-2 virus infection and the symptoms and related immune responses of clinical patients. Therefore, researchers have constructed key animal models capable of being infected with SARS-CoV-2 virus through transgenic, gene knock-in, viral delivery and other technologies, which will ultimately be used to study the infection mechanism of SARS-CoV-2 virus, evaluate the effectiveness of vaccines and antiviral drugs, and further explore its pathogenic mechanism.

[0003] However, these models all have their own shortcomings and cannot effectively simulate the pathological progression of the novel coronavirus infecting the human body, as follows:

[0004] (1) Stable genetic transgenic mouse model. To construct a SARS-CoV-2 infected mouse model, the key is to express the human ACE2 gene. In the early days, due to technical limitations, transgenic mice could be constructed by directly injecting the hACE2 expression vector driven by a heterologous gene promoter into the embryo. The promoters involved include mACE2, hK18, hHFH4 / FOXJ1, and CAG. The expression of the hACE2 gene driven by the mACE2 promoter is closer to the distribution of natural ACE2 in mice, but the expression of the hACE2 gene is weak. The expression of the hACE2 gene driven by the hK18 and hHFH4 / FOXJ1 human promoters is stronger, but it changes the distribution of ACE2 in mouse tissues, thereby changing the tissue tropism of the virus. In short, the disadvantage of this method is that the exogenously introduced hACE2 gene is randomly inserted into the mouse genome, which may interfere with the expression of other genes. At the same time, it cannot take into account the high expression of hACE2 in vivo and the expression distribution close to the actual situation in vivo. With the continuous maturity of CRISPR / Cas9 technology, this technology has also been used to construct transgenic mouse models and successfully solve the above two problems. The researchers used the CRISPR / Cas9 system to knock the hACE2 gene into exon 2 of the mouse endogenous ACE2 site. This method not only destroyed the expression of the mouse endogenous mACE2 gene, but also ensured that the expression distribution of the exogenous hACE2 gene was consistent with the expression of endogenous ACE2. In addition, by combining tetraploid compensation technology with CRISPR / Cas9 embryonic stem cell gene editing technology, ACE2 humanized inbred mice can be successfully established within 35 days. Although ACE2 was successfully humanized, the main symptom in mice was weight loss, and there was no way to better simulate human clinical symptoms.

[0005] (2) Mouse model transduced with Ad5-hACE2 or AAV-hACE2. In addition to overexpressing the hACE2 gene through genetically modified transgenic mice, ectopic expression of ACE2 can also be achieved in mice through virus-mediated gene delivery. Commonly used viral vectors include Adv or AAV. For example, through intranasal or tracheal inoculation, the adenovirus Ad5-hACE2 overexpressing hACE2 infects most lung epithelial cells, promoting the reproduction and replication of SARS-CoV-2 in the respiratory tract of mice. This process can last for several days, causing the mice to lose about 20% of their weight. During the infection, pathological changes such as inflammatory infiltration, hemorrhage, and alveolar edema can be observed in the lungs, indicating that lung function is impaired. Inflammatory factors such as TNF-α and IL-6 are also significantly upregulated. Compared with adenovirus, adeno-associated virus has lower immunogenicity. hACE2 is expressed in mouse lungs mediated by AAV, and then infected with SARS-CoV-2. The virus can also replicate in mouse lung tissue, thereby triggering increased infiltration of pulmonary monocytes, neutrophils and lymphocytes, and inducing type I interferon and humoral immune responses. Currently, the commonly used AAV serotypes for targeting mouse lung tissue are AAV-6 and AAV-9. Through intraperitoneal injection and tracheal inoculation of AAV-ACE2 virus, ACE2 can be continuously expressed in mouse lungs for up to 28 weeks. After SARS-CoV-2 virus attack, wild-type mice infected with AAV-ACE2 continued to replicate in the lungs of hACE2 mice for about 7 days, and moderate interstitial pneumonia symptoms appeared. These symptoms include peribronchial inflammation, diffuse infection of alveolar epithelium, infiltration of monocytes and derived macrophages, and different strains of mice have different immune responses. Due to the short-term expression of AAV and AV viruses, the humanization time of ACE2 gene is relatively short, the replication of the new coronavirus in the body is weak, and the corresponding clinical symptoms are also relatively mild, which has certain limitations in simulating the pathological characteristics of SARS-CoV-2 infection.

[0006] (3) Non-human primate model. Compared with mice, non-human primates (NHP), including rhesus monkeys, cynomolgus monkeys, African green monkeys, baboons and common marmosets, have significant similarities with humans in terms of physiological characteristics and immune regulation, so they can be used as large animal models of SARS-CoV-2 infection. Sequence alignment analysis of the ACE2 gene in multiple species showed that the homology between the ACE2 gene of the rhesus monkey and the human ACE2 gene was 91%, and the amino acid sequence of the key binding region RBD of ACE2 and S protein was conserved in humans, cynomolgus monkeys and rhesus monkeys. Despite this, different species of monkeys have different susceptibility to SARS-CoV-2, with African green monkeys being the most sensitive, followed by rhesus monkeys, and then cynomolgus monkeys. Although monkeys show typical clinical symptoms of human patients: lung discoloration, congestion, glassy opacities, infiltration, hemorrhage, necrosis and interstitial pneumonia, they are unable to reproduce the symptoms of clinically critically ill patients.

[0007] In general, all three NHPs are susceptible to SARS-CoV-2, but the symptoms are not as severe as those in clinical practice. Sequence analysis shows that the affinity of monkey ACE2 to RBD is lower than that of humans, which to some extent explains why NHPs can be infected with SARS-CoV-2 but do not develop severe symptoms. Therefore, ectopic expression of hACE2 in monkey lungs through AAV virus may be the best way to simulate the severe clinical symptoms caused by human SARS-CoV-2. Summary of the invention

[0008] The purpose of the present invention is to provide a method for rapidly preparing a humanized monkey model, to solve the current lack of a suitable novel coronavirus model, to better simulate the clinically relevant symptoms of patients, and to provide a better animal model for evaluating novel coronavirus vaccines and drugs.

[0009] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0010] A method for rapidly preparing a humanized monkey model comprises the following steps:

[0011] S1: Construct AAV9-ACE2-GFP vector, observe GFP expression, and detect ACE2 expression in infected cells by flow cytometry;

[0012] S2: Directly infect the mouse lungs with AAV9-ACE2 virus and detect the RNA expression level of human ACE2 to verify the expression effect of AAV9-ACE2 virus in mice;

[0013] S3: Target the AAV-ACE2 virus to the monkey lung organs and detect the expression of the human ACE2 gene in the lung tissue by Q-PCR;

[0014] S4: Use the humanized monkey model obtained in step S3 to conduct a novel coronavirus infection experiment.

[0015] Furthermore, the step S1 specifically includes the following sub-steps:

[0016] S1.1: Connect green fluorescent protein (GFP) as an indicator gene behind the ACE2 gene to construct an AAV9 vector capable of expressing the human ACE2 gene and GFP.

[0017] S1.2: A549 lung cancer cell line was inoculated in a 6-well plate, 2 mL of DMEM complete medium (containing 10% fetal bovine serum) was added to each well, and cultured for 24-36 hours until the cell confluence reached 50%.

[0018] S1.3: Add 2E+11GC and 8E+11GC AAV9-ACE2-GFP viruses respectively and infect for 48 hours.

[0019] S1.4: Use a fluorescence microscope to take pictures to observe the expression of GFP, and combine flow cytometry to detect the expression of ACE2 in infected cells.

[0020] Furthermore, the step S2 specifically includes the following sub-steps:

[0021] S2.1: Use endotracheal intubation to infect the mouse lungs with AAV9-ACE2 virus. After anesthesia, draw 200 μL of AAV9-ACE2 virus (titer 1E+13 GC / mL) from each mouse through a microsyringe and inject it into the mouse lungs.

[0022] S2.2: 14 days after infection, tissues were fixed by cardiac perfusion with saline and 4% paraformaldehyde, and then embedded in OCT and sectioned.

[0023] S2.3: The mouse lung tissue was removed for fluorescence microscopy observation of GFP expression, and the RNA expression level of human ACE2 was detected by RT-PCR to verify the expression effect of AAV9-ACE2 virus in mice.

[0024] Furthermore, the step S3 specifically includes the following sub-steps:

[0025] S3.1: First anesthetize the monkey and use a medical laryngoscope to assist in inserting a tracheal tube.

[0026] S3.2: Insert the medical laryngoscope into the medical laryngeal anesthesia sprayer, connect the sprayer to the syringe, and inject 1 ml of AAV9-ACE2 virus (titer: 1E+13GC / ml);

[0027] S3.3: After 3 months, the monkey’s lung tissue was removed, and RNA was extracted from different areas of the left and right lung lobes. The expression level of the human ACE2 gene in the lung tissue was detected by Q-PCR.

[0028] Furthermore, the step S4 specifically includes the following sub-steps:

[0029] S4.1: The humanized monkey model was infected by nasal drops and endotracheal intubation using the EG.5.1 variant of the novel coronavirus. The infection dose for each monkey was 1×10 7 TCID 50 .

[0030] S4.2: 7 days after infection, serum was collected on D0 and stored at -80°C for future use. Body temperature and body weight were measured 1 day after infection (1 dpi), 3 dpi, 5 dpi, and 7 dpi, and 2 nasal swabs, pharyngeal swabs, and anal swabs were collected and stored at -80°C for future use.

[0031] S4.3: Seven days after infection, all animals were euthanized and five samples of trachea, lungs (seven lung lobes were collected separately), tonsils, heart, liver, spleen, lungs, kidneys, brain (cerebrum and cerebellum), duodenum, jejunum, ileum, cecum, rectum, colon, and feces were collected and stored at -80°C. The remaining tissues were fixed in formalin and stored for later use.

[0032] Beneficial effects of the present invention:

[0033] The present invention adopts AAV9-ACE2 viral vector technology, and successfully expresses the human ACE2 gene ectopically in the humanized monkey model by directional injection of AAV9-ACE2 virus into the monkey lungs. The AAV9 viral vector has a high degree of lung targeting, and can effectively deliver the human ACE2 gene to monkey lung cells and achieve ectopic expression of the gene. In this process, the expression of the gene can be visually observed by using green fluorescent protein (GFP) as an indicator gene. Compared with traditional transgenic or gene knock-in technology, AAV9-ACE2 viral vector technology can quickly construct a humanized monkey model in a shorter period of time (about 3 months), greatly shortening the model preparation cycle and improving the efficiency and success rate of model construction.

[0034] In the present invention, the expression of human ACE2 gene in monkey lung tissue is quantitatively detected by Q-PCR technology to verify the expression effect of AAV9-ACE2 virus in the humanized monkey model. AAV9 virus has the characteristics of persistent and stable gene expression, and can maintain the efficient expression of human ACE2 gene for a long time. Combined with the precise quantification ability of Q-PCR technology, the efficient expression of human ACE2 gene in monkey lungs can be ensured. This efficient gene expression and quantitative detection technology not only verifies the successful construction of the model, but also provides reliable data support for subsequent experimental studies, ensuring the accuracy and repeatability of the model.

[0035] The present invention expresses the hACE2 gene in a humanized monkey model, and then uses the novel coronavirus EG.5.1 variant to conduct infection experiments in order to more realistically simulate the clinical symptoms of humans infected with SARS-CoV-2. During the model construction process, the AAV9-ACE2 virus is injected into the monkey's lungs through tracheal intubation technology. The virus has a high degree of lung targeting, ensuring that the human ACE2 gene is efficiently expressed in the lungs. The highly expressed human ACE2 gene significantly increases the susceptibility of monkey lung cells to SARS-CoV-2, and the virus can efficiently enter cells and reproduce, leading to typical lung pathological changes and clinical symptoms. These symptoms include lung inflammation, tissue infiltration, hemorrhage and necrosis, which simulate the pathological progression of human patients. In addition, through the dynamic monitoring of body temperature, weight, nasal swabs, pharyngeal swabs and anal swabs, the physiological changes during the infection process can be fully recorded, providing a more suitable animal model for the study of the novel coronavirus, which is helpful for in-depth exploration of the infection mechanism and pathogenic mechanism of the virus.

[0036] The humanized monkey model constructed by the present invention shows significant advantages in the evaluation of vaccines and antiviral drugs. By efficiently expressing the human ACE2 gene in the monkey's lungs, the effects of vaccines and drugs in the human body can be truly reflected, especially in terms of pathological changes and immune responses caused by viral infection. Using this model, dynamic monitoring of various time points after infection with the new coronavirus can be performed, including the detection of inflammatory factors and the evaluation of immune responses, so as to comprehensively evaluate the protective efficacy, immune response and safety of vaccines and drugs. Compared with the traditional mouse model, the humanized monkey model of the present invention has a higher similarity with humans in terms of physiological characteristics and immune regulation, and can more accurately predict the performance of vaccines and drugs in humans, greatly improving the efficiency and accuracy of vaccine and drug research and development.

[0037] In summary, the present invention uses AAV9-ACE2 viral vector technology to quickly construct a humanized monkey model, achieve efficient expression and quantitative detection of human ACE2 genes, effectively simulate the clinical symptoms of novel coronavirus infection, and provide an efficient and reliable model for the evaluation of vaccines and drugs. This technical solution not only improves research efficiency, but also provides strong support for the prevention and control of novel coronavirus.

[0038] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0040] Figure 1 Schematic diagram of the plasmid map of AAV-ACE2;

[0041] Figure 2 Schematic diagram of successful infection of A549 cells with AAV-ACE2-GFP;

[0042] Figure 3 Schematic diagram of the successful humanized mouse model of AAV-ACE2-GFP;

[0043] Figure 4 Schematic diagram of the detection of AAV neutralizing antibodies in cynomolgus monkeys;

[0044] Figure 5 This is a schematic diagram of the detection of ACE2 expression in cynomolgus monkeys;

[0045] Figure 6 Schematic diagram of tissue viral load detection in three humanized ACE2 crab-eating macaques. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1

[0048] The method for rapidly preparing a humanized monkey model described in this embodiment comprises the following steps:

[0049] 1. Construct an AAV9 vector expressing ACE2 and package it into a virus for verification at the cell level ( Figure 1 ).

[0050] In order to indicate the expression of ACE2 gene, GFP was expressed after ACE2 gene, and A549 lung cancer cell line was used as target cells and seeded in 6-well plates. When the cells grew to 50% confluence, 2E+11GC and 8E+11GC AAV viruses were added respectively. After 48h of infection, fluorescence microscopy was used to take pictures and flow cytometry was used to detect the positive rate of ACE2 expression in infected cells.

[0051] 2. Human ACE2 was successfully expressed in mice. Figure 1 and Figure 2 ).

[0052] In order to successfully express human ACE2 in mouse lungs, two steps were included: (1) AAV9-ACE2 virus was directed into the mouse lungs using endotracheal intubation. After 14 days of infection, the lung tissue was fixed by cardiac perfusion with saline and 4% paraformaldehyde, and then embedded in OCT and sliced, and the lung tissue was removed for subsequent testing. (2) The intensity of GFP expression in the tissue and the RNA expression level of human ACE2 were used to identify the effectiveness of AAV9-ACE2 expression.

[0053] 3. The AAV-ACE2 virus is targeted to the monkey's lung organs by tracheal intubation.

[0054] (1) First, the monkey was anesthetized by injecting anesthetics, and a medical laryngoscope was used to assist in inserting a medical laryngeal anesthesia tube sprayer. The sprayer was connected to a syringe and 1 ml of AAV9-ACE2 virus (titer: 1E+13GC / ml) was injected into it. (2) After 3 months, the monkey's lung tissue was obtained and divided into different areas of the left and right lung lobes. RNA was extracted separately, and Q-PCR was used to detect the expression of human ACE2 gene in the lung.

[0055] 4. The humanized monkey model was successfully infected with the EG.5.1 variant.

[0056] (1) Using the novel coronavirus EG.5.1 variant, the dose is: 1×10 7 TCID 50 / , infected humanized monkey model by nasal drops and endotracheal intubation. (2) Monkeys were killed 7 days after infection and tissue viral loads were tested in major organs. During the infection process, nasal swabs, pharyngeal swabs, and anal swabs were used to test the number of viral copies.

[0057] Example 2

[0058] The method for rapidly preparing a humanized monkey model described in this embodiment comprises the following steps:

[0059] Establishment of a cell line expressing human ACE2 ( Figure 1 ).

[0060] The method for overexpressing the human ACE2 gene by AAV virus is as follows: the human lung cancer epithelial cell line A549 is inoculated in a 6-well plate, with 2 mL of DMEM complete medium (10% FBS) in each well. After culturing for 24-36 hours, the cell confluence reaches 50%, and 2E+11GC and 8E+11GC AAV9-ACE2-GFP viruses are added respectively. After 48 hours of infection, the GFP positivity rate in the cells is detected.

[0061] Establishment of humanized mouse ACE2 model ( Figure 2 ).

[0062] The method for establishing a humanized ACE2 mouse model using endotracheal intubation technology is as follows: 100 μL of anesthetic is injected intraperitoneally into each mouse before surgery, and 200 μL of AAV virus (titer: 1E+13 GC / mL) is drawn up using a microsyringe and injected into the mouse lungs. Three weeks after infection, a portion of the mouse lung tissue is taken for tissue staining after standard cardiac perfusion, and a portion is used to extract RNA to detect the expression of the human ACE2 gene.

[0063] Establishment of humanized ACE2 monkey model ( Figure 2 ).

[0064] The method for establishing a humanized ACE2 mouse model using endotracheal intubation technology is as follows: each monkey is intravenously injected with a corresponding dose of anesthetic before surgery, and 1 mL of AAV virus (titer: 1E+13GC / mL) is drawn using a microsyringe and injected into the monkey's lungs. Three months after infection, the mouse lung tissue is obtained after standard cardiac perfusion for RNA extraction and detection of human ACE2 gene expression.

[0065] Humanized ACE2 monkey model infected with novel coronavirus ( Figure 2 ).

[0066] Three model monkeys were challenged with the virus in a laboratory qualified to carry out the novel coronavirus. Three months after the monkeys were infected with the AAV-ACE2 virus, they were infected with the novel coronavirus EG.5.1 variant by nasal drops and tracheal intubation at a dose of 1×10 7 TCID 50 / , 7 days after the virus attack, serum was collected on D0 and stored at -80℃ for later use. Body temperature and weight were measured 1 day after the virus attack (1dpi), 3dpi, 5dpi, and 7dpi, and 2 nasal swabs, pharyngeal swabs, and anal swabs were collected and stored at -80℃ for later use. All animals were euthanized seven days after the virus attack, and 5 samples of trachea, lung (7 lobes of each lung were collected), tonsils, heart, liver, spleen, lung, kidney, brain (cerebrum and cerebellum), duodenum, jejunum, ileum, cecum, rectum, colon, and feces were collected and stored at -80℃, and the remaining tissues were fixed with formalin.

[0067] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for rapidly preparing a humanized monkey model, characterized in that: The following steps are involved: S1: Construct AAV9-ACE2-GFP vector, observe GFP expression, and detect ACE2 expression in infected cells by flow cytometry; S2: Directly infect the mouse lungs with AAV9-ACE2 virus and detect the RNA expression level of human ACE2 to verify the expression effect of AAV9-ACE2 virus in mice; S3: Target the AAV-ACE2 virus to the monkey lung organs and detect the expression of the human ACE2 gene in the lung tissue by Q-PCR; S4: Use the humanized monkey model obtained in step S3 to conduct a novel coronavirus infection experiment.

2. The method for rapidly preparing a humanized monkey model according to claim 1, characterized in that: The step S1 specifically includes the following sub-steps: S1.1: Connect green fluorescent protein as an indicator gene behind the ACE2 gene to construct an AAV9 vector that can express the human ACE2 gene and GFP; S1.2: Inoculate A549 lung cancer cell line in a 6-well plate, add 2 mL of DMEM complete medium to each well, and culture for 24-36 hours until the cell confluence reaches 50%; S1.3: Add 2E+11GC and 8E+11GC AAV9-ACE2-GFP viruses, respectively, and infect for 48 hours; S1.4: Use a fluorescence microscope to take pictures to observe the expression of GFP, and combine flow cytometry to detect the expression of ACE2 in infected cells.

3. The method for rapidly preparing a humanized monkey model according to claim 1, characterized in that: The step S2 specifically includes the following sub-steps: S2.1: Use endotracheal intubation to infect the mouse lungs with AAV9-ACE2 virus; after anesthesia, draw 200 μL of AAV9-ACE2 virus from each mouse through a microsyringe and inject it into the mouse lungs; S2.2: 14 days after infection, tissues were fixed by cardiac perfusion with saline and 4% paraformaldehyde, and then embedded in OCT and sectioned; S2.3: The mouse lung tissue was removed for fluorescence microscopy observation of GFP expression, and the RNA expression level of human ACE2 was detected by RT-PCR to verify the expression effect of AAV9-ACE2 virus in mice.

4. The method for rapidly preparing a humanized monkey model according to claim 1, characterized in that: The step S3 specifically includes the following sub-steps: S3.1: First, anesthetize the monkey and insert the endotracheal tube using a medical laryngoscope; S3.2: Insert the medical laryngoscope into the medical laryngeal anesthesia sprayer, connect the sprayer to the syringe, and inject 1 ml of AAV9-ACE2 virus into it; S3.3: After 3 months, the monkey’s lung tissue was removed, and RNA was extracted from different areas of the left and right lung lobes. The expression level of the human ACE2 gene in the lung tissue was detected by Q-PCR.

5. The method for rapidly preparing a humanized monkey model according to claim 1, characterized in that: The step S4 specifically includes the following sub-steps: S4.1: The humanized monkey model was infected by nasal drops and endotracheal intubation using the EG.5.1 variant of the novel coronavirus; the infection dose for each monkey was 1×10 7 TCID 50 ; S4.2: 7 days after infection, serum was collected on D0 and stored at -80℃ for future use. Body temperature and body weight were measured at 1dpi, 3dpi, 5dpi, and 7dpi after infection, and 2 nasal swabs, pharyngeal swabs, and anal swabs were collected and stored at -80℃ for future use. S4.3: Seven days after infection, all animals were euthanized and five samples of trachea, lungs, tonsils, heart, liver, spleen, lungs, kidneys, brain, duodenum, jejunum, ileum, cecum, rectum, colon and feces were collected and stored at -80°C. The remaining tissues were fixed in formalin and stored for later use.

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