Method for establishing dengue hemorrhagic fever animal model for expressing hTim4

Abstract

The invention discloses a method for establishing a dengue hemorrhagic fever animal model for expressing hTim4, and belongs to the technical field of transgenic animal models. The establishment method of the dengue hemorrhagic fever animal model expressing the hTim4 comprises the following steps: transferring a vector containing a DNA fragment of the human-derived Tim4 into a mouse fertilized egg, carrying out transgenosis treatment on the mouse by using a Tol2 transgenosis system, implanting the survived fertilized egg into a false pregnancy receptor mouse, screening to obtain a positive mouse highly expressing the hTim4, and carrying out screening to obtain the dengue hemorrhagic fever animal model expressing the hTim4. And injecting dengue virus into the positive mouse intracranial. The clinical characteristics of the dengue hemorrhagic fever animal model expressing hTim4 highly conform to the clinical manifestation of human dengue hemorrhagic fever, and the model can be widely applied to the research of dengue hemorrhagic fever and the development of new drugs.

Description

Technical Field

[0001] The invention relates to the technical field of transgenic animal models, and in particular to a method for establishing a dengue hemorrhagic fever animal model expressing hTim4. Background Art

[0002] Dengue fever, caused by dengue virus (DENV), is considered one of the most important arthropod-borne viral infections affecting people in tropical and subtropical regions. Currently, approximately one-third of the global population is at risk of DENV infection, with an estimated 400 million infections each year. The continued spread of dengue fever around the world can be attributed to abrupt climate change, rapid and unplanned urbanization and construction, high population density, and ineffective vector control strategies. The scientific community must increase its attention to addressing this ongoing dengue epidemic. In particular, in 2023, more than 5 million infections were reported worldwide, including 5,000 deaths, close to the historical peak. In addition, new regions around the world have been affected, including southern Europe. In addition, as of April 2, 2024, dengue virus has been reported to have caused more than 1 million infections and multiple deaths in Brazil and Argentina, among others.

[0003] There are four common serotypes of dengue infection, namely DENV-1 to DENV-4. A new serotype, DENV-5, was discovered in Malaysia in 2007. Individuals infected with dengue virus can be asymptomatic or develop mild dengue fever, neurological complications, or severe symptoms such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which can be life-threatening. Severe dengue fever is characterized by systemic bleeding tendency, thrombocytopenia, capillary leakage, organ failure, shock, and occasionally death. The severe clinical symptoms of DHF / DSS caused by dengue virus infection are manifested as high fever, high viremia, severe bleeding, cytokine storm, hypovolemic shock, and multiple organ dysfunction failure. In DENV secondary heterotypic infection, the circulation of specific IgG antibodies activates the complement system that mediates vascular permeability. The cross-reaction of antibody immune complexes Fc receptors formed by DENVNS1 viral protein with vascular endothelial cells (Cross-react with ECs) enhances vascular permeability. Monocytes, macrophages, mast cells, and immune B and T cells respond to DENV pathogen infection by producing cytokines to inhibit viral replication and even induce cytokine storms that lead to increased vascular permeability.

[0004] Vascular permeability changes are a hallmark event of severe dengue infection. Endothelial cells line blood vessels and capillaries, forming a highly selective physical barrier. These ECs are central elements of tissue and organ metabolism and physiology. DENV can directly infect ECs, triggering apoptosis, increasing vascular permeability, etc., leading to organ dysfunction. Viral antigens, morphological changes, and capillary inflammation were detected in postmortem biopsies of severe dengue cases. These changes were associated with mononuclear cell membrane permeability, edema, inflammation, and perivascular cell accumulation. The susceptibility of these cells to the virus has also been confirmed in vitro, however, this susceptibility does not explain the endothelial dysfunction or severe tissue and organ systemic disease events that occur during infection. Endothelial damage is a key indicator of disease severity and potential lethality in dengue infection. DENV is affected during infection, where the uncontrolled response of the immune system and the elevated concentrations of some cytokines are crucial for endothelial damage. Currently, due to the lack of an animal disease model that effectively simulates dengue hemorrhagic fever, the clear mechanism of severe bleeding symptoms remains to be revealed and confirmed.

[0005] The mouse model has unique comprehensive advantages among model organisms, not only in terms of genetics and physiology, but also in terms of low cost, fast and convenient breeding and rearing, clear genetic background, and mature corresponding gene editing technology, making it the most important model animal for disease models and drug development and evaluation research in the field of emerging infectious diseases. The construction of mouse disease models can help us deeply reveal the pathogenic mechanism of intestinal bleeding, provide tools for preclinical evaluation research, and is an important bridge for the effective transformation of basic research in drug development to clinical prevention and treatment applications. However, there is currently a lack of a model animal that can be used for the pathogenic mechanism of dengue hemorrhagic fever. Summary of the invention

[0006] In response to the above-mentioned problem of the current lack of model animals that can be used to accurately evaluate dengue hemorrhagic fever, the present invention provides a method for establishing an animal model of dengue hemorrhagic fever expressing hTim4. The model animal of dengue hemorrhagic fever established by this method can simulate the typical clinical characteristics of dengue hemorrhagic fever, and can be used in the research and treatment of the pathogenic mechanism of intestinal bleeding, especially in the development and evaluation of drugs for dengue hemorrhagic fever.

[0007] On the one hand, the present invention discloses a method for establishing an animal model of dengue hemorrhagic fever expressing hTim4, comprising the following steps: transferring a vector containing a DNA fragment of human Tim4 into a mouse fertilized egg, subjecting the mouse to transgenic treatment using a Tol2 transgenic system, and implanting the surviving fertilized eggs into pseudo-pregnant recipient mice, obtaining positive mice highly expressing hTim4 through screening, and injecting the dengue virus intracranially into the positive mice.

[0008] In some of the schemes, the method for establishing an animal model of dengue hemorrhagic fever expressing hTim4 comprises the following steps:

[0009] Targeting vector construction: Construction of a targeting vector containing a DNA fragment of human Tim4;

[0010] Cultivate F0 generation mice: inject the positive targeting vector and the vector encoding Tol2 enzyme into the fertilized egg cells of mice, and transplant the surviving fertilized eggs into pseudo-pregnant mice, and select the positive mice among the born mice, which are the F0 generation mice for future use;

[0011] Breeding: The F0 generation mice were mated with wild-type mice of the same strain, and the mice expressing hTim4 were obtained as positive mice;

[0012] Infection: Inject dengue virus into the positive mice intracranially to obtain the dengue hemorrhagic fever animal model.

[0013] In some of the schemes, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-WPRE. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-WPRE-PolyA.

[0014] It can be understood that the above-mentioned CAG, KOZAK, P2A, WPRE, PolyA and other sequence fragments play the roles of promoter of gene expression, improving protein translation, protein cleavage, enhancing target gene expression and increasing RNA stability through post-transcriptional regulatory mechanism, improving translation efficiency, etc. Different functional gene fragments can also be selected according to the common usage in the field, and it is only necessary to ensure that the hTim4 gene subsequently transferred can be stably inherited and overexpressed.

[0015] In some of the schemes, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP-WPRE. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP-WPRE-PolyA.

[0016] It can be understood that the above-mentioned EGFP sequence fragment can encode environmentally sensitive green fluorescent protein, which can be used as a marker for subsequent positive mouse screening. From the perspective of the function of the model itself, it is not necessary.

[0017] In some of the embodiments, the DNA sequence of hTim4 is shown as SEQ ID NO:1.

[0018] In some embodiments, the sequence of CAG is shown as SEQ ID NO:2.

[0019] In some of the embodiments, the sequence of KOZAK is gccacc.

[0020] In some of the embodiments, the sequence of P2A is shown as SEQ ID NO:3.

[0021] In some of the embodiments, the sequence of EGFP is shown as SEQ ID NO:4.

[0022] In some embodiments, the sequence of the WPRE is shown as SEQ ID NO:5.

[0023] In some of the embodiments, the sequence of PolyA is shown as SEQ ID NO:6.

[0024] In some of the embodiments, the targeting vector sequence is shown as SEQ ID NO:7.

[0025] In some embodiments, the dengue virus is dengue virus type 2.

[0026] In some embodiments, the vector is a Tol2 vector.

[0027] It can be understood that the above-mentioned Tol2 vector is also called a transposon plasmid, which contains two inverted terminal repeat sequences (ITRs) and a transposition region therebetween. The human Tim4 gene is cloned in this region, and the rest of the design can refer to conventional design.

[0028] In some of these protocols, the mice are C57BL / 6 mice.

[0029] In some of the embodiments, the Tol2 mRNA sequence encoding the Tol2 enzyme is shown in SEQ ID NO:8.

[0030] In some of the embodiments, the vector sequence encoding the Tol2 enzyme is shown as SEQ ID NO:15.

[0031] In some of these schemes, the primer pair shown as SEQ ID NO:9-SEQ ID NO:10 is used to screen positive mice.

[0032] In some of the schemes, the targeting vector contains an EGFP sequence fragment, and positive mice are screened by in vivo fluorescence detection using a CT imaging system or by using a primer pair as shown in SEQ ID NO:11-SEQ ID NO:12.

[0033] On the other hand, the present invention also discloses a method for establishing a dengue hemorrhagic fever susceptible animal model expressing hTim4, comprising the following steps: transferring a vector containing a DNA fragment of human Tim4 into a mouse fertilized egg, subjecting the mouse to transgenic treatment using the Tol2 transgenic system, and implanting the surviving fertilized eggs into pseudo-pregnant recipient mice, and obtaining positive mice with high expression of hTim4 through screening.

[0034] In some of the schemes, the method for establishing a dengue hemorrhagic fever susceptible animal model expressing hTim4 comprises the following steps:

[0035] Targeting vector construction: Construction of a targeting vector containing a DNA fragment of human Tim4;

[0036] Cultivate F0 generation mice: inject the positive targeting vector and the vector encoding Tol2 enzyme into the fertilized egg cells of mice, and transplant the surviving fertilized eggs into pseudo-pregnant mice, and select the positive mice among the born mice, which are the F0 generation mice for future use;

[0037] Breeding: The F0 generation mice are mated with wild-type mice of the same strain, and the mice expressing hTim4 are obtained as positive mice.

[0038] In some of the schemes, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-WPRE. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-WPRE-PolyA.

[0039] In some of the schemes, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP-WPRE. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP-WPRE-PolyA.

[0040] In some of the embodiments, the DNA sequence of hTim4 is shown as SEQ ID NO:1.

[0041] In some embodiments, the sequence of CAG is shown as SEQ ID NO:2.

[0042] In some of the embodiments, the sequence of KOZAK is gccacc.

[0043] In some of the embodiments, the sequence of P2A is shown as SEQ ID NO:3.

[0044] In some of the embodiments, the sequence of EGFP is shown as SEQ ID NO:4.

[0045] In some embodiments, the sequence of the WPRE is shown as SEQ ID NO:5.

[0046] In some of the embodiments, the sequence of PolyA is shown as SEQ ID NO:6.

[0047] In some of the embodiments, the targeting vector sequence is shown as SEQ ID NO:7.

[0048] In some embodiments, the vector is a Tol2 vector.

[0049] In some of these protocols, the mice are C57BL / 6 mice.

[0050] In some of the embodiments, the Tol2 sequence encoding the Tol2 enzyme is shown as SEQ ID NO:8.

[0051] In some of the embodiments, the vector sequence encoding the Tol2 enzyme is shown as SEQ ID NO:15.

[0052] In some of these schemes, the primer pair shown in SEQ ID NO:9-SEQ ID NO:10 is used to screen positive mice;

[0053] In some of the schemes, the targeting vector contains an EGFP sequence fragment, and positive mice are screened by in vivo fluorescence detection using a CT imaging system or by using a primer pair as shown in SEQ ID NO:11-SEQ ID NO:12.

[0054] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0055] The reagents and raw materials used in the present invention are commercially available.

[0056] The positive and progressive effects of the present invention are:

[0057] The present invention discloses a method for establishing an animal model of dengue hemorrhagic fever expressing hTim4. The humanized Tim4 gene is transgenic edited in mice through the Tol2 transgenic system. The obtained mouse model can have stable heredity. The model overexpresses humanized Tim4 (hTim4). The model has normal immune function and is highly susceptible to DENV-2 infection. After infection with dengue virus 2, typical DHF / DSS characteristics such as systemic bleeding, neurological complications, severe intestinal dysfunction and death can be simulated. More importantly, a Th2-type biased cytokine storm consistent with human response was observed in the model. These findings provide the first convincing in vivo evidence supporting Tim4 as a functional receptor for dengue virus, and the model can be widely used in the research and new drug development of dengue hemorrhagic fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of the targeting vector structure.

[0059] Figure 2 The gel electrophoresis diagram of the products verified by enzyme digestion and linearization.

[0060] Figure 3 Schematic diagram of electrophoresis bands for amplification identification.

[0061] Figure 4 Schematic diagram of electrophoresis bands for identifying positive mice in the F1 generation.

[0062] Figure 5 The EGFP fluorescence photos of mice were identified by IVIS Spectrum CT imaging system.

[0063] Figure 6 is the expression level of hTim4 in various tissues of mice.

[0064] Figure 7 This is the in vitro complex result of DENV-2 and hTim4.

[0065] Figure 8These are the semi-in vitro adsorption results of primary spleen cells and hepatocytes with DENV-2.

[0066] Fig. 9 DENV-2 proliferation in primary splenocytes and semi-ex vivo propagated hepatocytes.

[0067] Fig.10 Results of RNA-seq test and cell flow cytometry analysis.

[0068] Fig.11 It is the result of coagulation function parameter index.

[0069] Fig.12 This is the viral copy status after mice were infected with DENV-2.

[0070] Fig.13 This is a statistical chart of the survival period of mice infected with DENV-2.

[0071] Fig.14 This is a statistical chart of weight changes in mice after infection with DENV-2.

[0072] Fig.15 This is a graph showing the clinical symptom scores of mice infected with DENV-2.

[0073] Fig.16 This shows the viral load in multiple organ tissues of mice after infection with DENV-2.

[0074] Fig.17 This is a picture of multiple organ bleeding after mice were infected with DENV-2.

[0075] Fig.18 This is the HE staining image of brain pathology in hTim4 model mice.

[0076] Fig.19 HE staining of lung pathology in hTim4 model mice.

[0077] Fig. 20 This is the HE staining image of intestinal pathology in hTim4 model mice.

[0078] Fig.21 Schematic diagram of the coagulation status of hTim4 model mice.

[0079] Fig. 22 Schematic diagram of prothrombin time and partial prothrombin activation time in hTim4 model mice.

[0080] Fig.23 This is a picture of neutrophils and leukocytes in hTim4 model mice.

[0081] Fig.24 Schematic diagram of intestinal tissue bleeding, congestion, and even intestinal failure in hTim4 model mice.

[0082] Fig.25 This is an immunofluorescence staining image of hTim4 model mice.

[0083] Fig.26 This is the spatial transcriptome sequencing map of mice.

[0084] Fig. 27 Schematic diagram of the comparison of cell types in intestinal tissues of the dengue virus DENV-2 infected group and the uninfected group.

[0085] Fig.28 This is the differential gene analysis between the dengue virus DENV-2 infected and uninfected groups in hTim4 model mice.

[0086] Fig.29 This is the interaction diagram between differentially expressed genes and cytokines in the dengue virus DENV-2 infected and uninfected groups of hTim4 model mice.

[0087] Fig.30 The differential gene expression levels between the dengue virus DENV-2 infected group and the uninfected group in hTim4 model mice.

[0088] Fig.31 This is a graph showing changes in cytokines in hTim4 model mice.

[0089] Fig.32 This is a graph showing changes in human cytokines. DETAILED DESCRIPTION

[0090] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0091] Experimental materials: SPF-grade C57BL / 6 mice were provided by the Institute of Animal Resources, China Food and Drug Administration. Laboratory Animal Production License No.: SCXK (Beijing) 2022-0002. Animals were kept in the SPF-grade animal room of the model animal department. All animals had free access to water and food and were subject to a 12-h light / dark cycle. All animal experiments were approved by the Laboratory Animal Committee of China Food and Drug Administration (approval numbers: 2019-B-16, 2024-B-027). The human cytokine detection process was reviewed by the Ethics Committee of the Yunnan Institute of Parasitic Diseases (No. 03). All animal experiments strictly adhered to the internationally recognized principles of laboratory animal care and use, and followed the ARRIVE guidelines and the American Veterinary Medical Association (AVMA) guidelines.

[0092] Example 1

[0093] A dengue hemorrhagic fever susceptible mouse model expressing hTim4 was established by the following method.

[0094] 1. Design Concept

[0095] After extensive research and analysis of the dengue infection process, the inventors believe that host receptors not only play a crucial role in determining cell susceptibility and serving as an entry point for viral infection of the body, but also play a pivotal role in vaccine screening, development and evaluation. Therefore, they decided to establish a transgenic susceptible animal model of dengue virus receptors.

[0096] Although various possible functional receptors for dengue virus, including AXL, CD14-associated protein, Claudin-1, C-type lectin domain family 5 member A (CLEC5A), CRD4-7-Fc, DC-SIGN / L-SIGN, Diverse, FcγR, GRP78, Heparan sulfate, Laminin receptor, HSPGs, HSP70 / 90, Integrinβ3, Mannose receptor (MR, CD206), nLc4Cer, prohibitin, Tim-1 / Tim-4 and TAM family proteins, have been proposed in the past few decades, there are still some controversies. However, it is worth noting that all these receptor candidates only show susceptibility to DENV in vitro, without any available in vivo evidence. The reason may be that it is difficult to establish a transgenic model animal that can be stably inherited, and there is no good animal model available in the industry.

[0097] Since Tim1 (T cell immunoglobμlin domain and mucin domain protein-1) is claimed to be a functional receptor for dengue virus entry because it can confer infectivity to non-susceptible cells, the present invention first considered constructing an animal model of Tim1 overexpression, but subsequent experiments using transgenic mice to overexpress Tim1 did not support DENV infection, which highlights the inadequacy of relying solely on in vitro evidence to confirm functional receptors. On this basis, the inventors, after many attempts, established transgenic mice overexpressing hTim4, allowing hTim4 to be stably inherited and overexpressed, and finally successfully established the first animal model of dengue susceptible cells with normal immune function.

[0098] 2. Establishment Method

[0099] 1. Construction of targeting vector

[0100] After analysis, the human Tim4 gene is located on chromosome 5 (Ensembl: ENSG00000145850), and the CDS fragment is 1246 bp in length. A targeting vector containing the structure of "CAG-KOZAK-hTim4-P2A-EGFP-WPRE-PolyA" was designed and constructed (such as Figure 1 The specific method is as follows:

[0101] The human Tim4 gene cDNA sequence (NM_001146726.2) was downloaded from NCBI and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and then digested with SwaI and AsiSI and connected to the Tol2 vector (Add Gene: Catalog No.: #22797).

[0102] Among them, the hTim4 sequence is:

[0103]

[0104] In the above-mentioned "CAG-KOZAK-hTim4-P2A-EGFP-WPRE-PolyA" structure, the sequence of CAG is shown in SEQ ID NO:2; the sequence of KOZAK is gccacc; the sequence of P2A is shown in SEQ ID NO:3; the sequence of EGFP is shown in SEQ ID NO:4; the sequence of WPRE is shown in SEQ ID NO:5; the sequence of PolyA is shown in SEQ ID NO:6; and the targeting vector sequence is shown in SEQ ID NO:7.

[0105] The specific steps of constructing the above-mentioned targeting vector are as follows:

[0106] 1) After the targeting vector containing the structure of "CAG-KOZAK-hTim4-P2A-EGFP-WPRE-PolyA" was synthesized, the target fragment was cut out using SwaI and AsiSI, and recovered by agarose gel. The Tol2 backbone was then cut with the same restriction endonuclease and recovered by agarose gel. Since the two had the same sticky ends, the target fragment was ligated to the Tol2 vector using T4 DNA ligase;

[0107] 2) Transformation: After the ligation reaction is completed, place the ligation product on ice for 2 minutes, cool it down, and directly add it to 100 μL Top10 competent cells. After transformation for 30 minutes, heat shock it for 90 seconds, place it on ice for 2 minutes, and finally add it to 600 μL SOC medium without antibiotics. Incubate it on a shaker at 37°C and 150 rpm for 1 hour, and then use a sterilized spreader to evenly spread the bacterial solution on a solid LB plate. Incubate it upside down at 37°C for 16 hours;

[0108] 3) Clone picking and shaking: Pick 4-6 single clones directly from the plate, inoculate them into 2.5 mL of ampicillin-resistant liquid LB medium, and incubate them in a shaking incubator at 37°C and 150 rpm for 12-16 hours;

[0109] 4) 200 μL of bacterial solution was kept at 4°C, and the remaining bacterial solution was collected and the plasmid was extracted according to the steps in the plasmid extraction kit (Takara, Code No. 9760);

[0110] 5) Enzyme digestion was performed using SwaI / AsiSI endonuclease and XbaI endonuclease, respectively, and the resulting fragment sizes were 1839bp / 7852bp and 2685bp / 7006bp;

[0111] 6) Sequencing confirmation of the verified correct plasmid;

[0112] 7) The plasmid solution with correct sequencing was extracted using the plasmid extraction kit (QIAGEN, Cat. No. 12362). After extraction, the enzyme digestion was verified to be consistent with the expectations. The gel electrophoresis of the enzyme digestion verification and linearization products was as follows: Figure 2 As shown in the figure, the samples in lanes 1, 2, and 3 represent the undigested plasmid control (original plasmid), the digestion product of endonuclease SwaI / AsiSI, and the digestion product of endonuclease XbaI, respectively, and lane M is a marker (GeneRμlerTM 1kb plus DNA Ladder, Thermo Fisher Company, #R0491).

[0113] According to the common knowledge in the art, a helper plasmid vector containing the Tol2 sequence (SEQ ID NO: 8) encoding the Tol2 enzyme was designed and prepared, and its sequence is shown in SEQ ID NO: 15.

[0114] 2. Cultivation of F0 generation mice

[0115] The mixture of the above-mentioned verified correct targeting vector and the vector encoding the Tol2 enzyme (such as SEQ ID NO: 15) was directly injected into the mouse fertilized egg cells by microinjection using an Eppendorf NK2 microinjector, and cultured in a 5% CO2, 37°C incubator for 24 hours. The viable fertilized egg cells were selected and transplanted into the oviducts of C57BL / 6 pseudo-pregnant female mice. A total of 103 fertilized eggs were transplanted into 4 pseudo-pregnant mice, and a total of 24 F0 mice were born. The genomic DNA of all mouse tails was extracted, and the positive mice were screened by PCR method using the following primer pairs.

[0116] Table 1. Genotype identification primers

[0117]

[0118]

[0119] The above primer pair F1 / R1 was used for PCR verification to verify that the targeting vector was correctly inserted into the human Tim4 sequence, and F2 / R2 was used to verify that the targeting vector was correctly inserted into the EGFP sequence.

[0120] Table 2. Genotyping PCR program

[0121]

[0122] Table 3. PCR reaction system

[0123]

[0124] Weigh 1g of agarose, heat and dissolve it in 1x TAE solution to prepare a 1% agarose gel solution. When the solution cools to 60-70°C, add 4S Red Plus (1μl / 10mL) and mix thoroughly. Pour the agarose solution into the gel mold and insert the tooth comb according to the number of samples. When the gel block solidifies, place the amplified product in the electrophoresis tank for electrophoresis. After the electrophoresis is completed, image and take pictures using a gel imager.

[0125] The identification results are as follows Figure 3 As shown, Figure 3 The upper figure is a schematic diagram of the electrophoresis band of the hTim4 gene amplified by the F1 and R1 primer pairs, and the lower figure is a schematic diagram of the electrophoresis band of the EGFP gene amplified by the F2 and R2 primer pairs. The results show that three positive mice (F0, 17#, 31#, 69#) were obtained in this experiment and named C57BL / 6-Tg(hTim4) / Nifdc model mice.

[0126] 3. Breeding

[0127] The hTim4 model mice obtained above are F0 generation positive mice. By mating them with wild-type C57BL / 6J mice, the F1 generation positive mice expressing hTim4 are model mice (hTim4 model mice) that can be stably inherited and used for maintaining the population and producing reproduction, that is, the dengue hemorrhagic fever susceptible mouse model expressing hTim4. The identification results are as follows Figure 4 As shown, the leftmost and rightmost lanes are markers, and the F1 generation mice showed 302 bp and 328 bp bands, indicating that they can stably express hTim4 and EGFP genes.

[0128] Example 2

[0129] This example verifies the hTim4 model mouse established in Example 1.

[0130] 1. Fluorescence detection

[0131] 1. Methods

[0132] Newborn positive mice, i.e. hTim4 model mice, aged 3 to 5 days were selected for in vivo fluorescence detection using IVIS Spectrum CT Imaging System. The expression of hTim4 was determined by EGFP immunofluorescence through radiant efficiency analysis.

[0133] 2. Results

[0134] The results are as follows Figure 5As shown in the figure, the identification of EGFP fluorescence by the IVIS spectral CT imaging system confirmed that the hTim4 gene can be systemically expressed in hTim4 model mice (labeled as hTim4, green on the figure), but not in newborn wild-type C57BL / 6 (labeled as WT, gray on the figure) mice.

[0135] 2. RT-qPCR detection

[0136] RT-qPCR was used to detect the expression level of hTim4 in different tissues at 6 weeks after birth.

[0137] 1. Methods

[0138] Weigh 0.1 g of fresh tissue of the target animal soaked in RNA stabilizing solution and add RNA rapid extraction reagent RNA isoplus (0.1g / 1mL), add RNase-free steel beads, use a pre-cooled tissue homogenizer to break the tissue until there are no large pieces of tissue visible to the naked eye; add 0.2mL pre-cooled chloroform, invert and mix, let stand on ice for 5 minutes, then centrifuge at 12000 rpm at 4°C for 5 minutes; take the upper aqueous phase, add 0.5mL pre-cooled isopropanol, invert and mix, let stand on ice for 10 minutes, then centrifuge at 12000 rpm at 4°C for 10 minutes; discard the supernatant, add 0.5mL pre-cooled 75% ethanol (anhydrous ethanol diluted with DEPC water) to wash the precipitate, and centrifuge at 7500 rpm at 4°C for 5 minutes; pour off the ethanol, dry at room temperature, dissolve the precipitate with DEPC water, and determine its concentration and purity by Nanodrop2000 ultraviolet spectrophotometer. The purity is 1.8-1.9. According to the concentration, the RNA sample can be used as a reverse transcription template after being diluted to 400ng / μl with DEPC water.

[0139] The reverse transcription experiment was performed in two steps using a reverse transcription kit (PrimeScript RT reagent Kit with gDNA Eraser; Takara). The reaction system for removing genomic DNA was configured according to the following Table 4. The primers used in the reaction were shown in Table 1. The PCR reaction procedure was: 42°C, 2 minutes, and stored at 4°C.

[0140] Table 4. Reaction system for removing genomic DNA

[0141]

[0142] After the genomic DNA is removed, the system described in Table 5 is added to the solution. The primers used for reverse transcription are shown in Table 1. The PCR reaction program is: 37°C, 15 minutes; 85°C, 5 seconds; 4°C storage. The tissue cDNA generated by the reaction can be used as a template for subsequent real-time quantitative polymerase chain reaction.

[0143] Table 5. Reverse transcription reaction system

[0144]

[0145] According to the RT-qPCR reaction system listed in Table 6, the qPCR primers are shown in Table 1. Each group requires 3 samples, each sample is set with 3 replicate wells, and each PCR plate is set with one identical sample replicate to exclude inter-plate differences. GAPDH is used as a control for relative quantitative PCR reaction. Pre-denaturation: 95°C for 30 seconds; enrichment: 95°C for 5 seconds, 65°C for 30 seconds, 40 cycles; melting curve: 95°C for 5 seconds, 65°C for 1 minute, 95°C 0.5°C (increase 0.5°C per unit time until 95°C); store at 4°C.

[0146] Table 6. Reverse transcription reaction system

[0147]

[0148] The primers are shown in Table 1.

[0149] 2. Results

[0150] The results are as follows Figure 6 As shown in the figure, the horizontal axis from left to right is heart, liver, spleen, kidney, intestine, brain, and genitals, and the vertical axis is the hTim4 expression level determined by RT-qPCR. The results showed that at week 6, hTim4 was significantly highly expressed in the heart, liver, spleen, kidney, intestine, brain, and genitals of the hTim4 model mouse group (hTim4) compared with the control mouse group (WT).

[0151] Example 3

[0152] Performance testing experiment of hTim4 model mice.

[0153] 1. Methods

[0154] 1. In vitro composite experiment

[0155] (1) Virus: 10 6 TCID 50 / ml DENV-2Tr1751 strain (from Capital Medical University).

[0156] (2) Protein: hTim4-Fc soluble protein (from Harbin Medical University).

[0157] (3) Experimental groups: DENV-2 group, DENV-2 mixed with different doses of hTim4-Fc soluble protein group.

[0158] (4) Experimental plan: 10 6 TCID 5010 μl of DENV-2Tr1751 strain at 4% / ml was mixed with 0.2 μg, 1 μg and 5 μg of hTim4-Fc soluble protein to form DENV-2 / hTim4 complex, which was purified with protein G agarose beads. RNA in the complex was then extracted for quantitative detection.

[0159] 2. Semi-isolated adsorption experiment

[0160] (1) Virus: Same as above.

[0161] (2) Primary cells: primary spleen cells and primary hepatocytes of wild-type C57BL / 6 and hTim4 model mice (obtained from primary culture of the hTim4 model mouse tissues mentioned above).

[0162] (3) Experimental groups: wild-type C57BL / 6 (WT) primary spleen cells 4℃ group, hTim4 model mouse primary spleen cells 4℃ group, wild-type C57BL / 6 (WT) primary hepatocytes 4℃ group, hTim4 model mouse primary hepatocytes 4℃ group, wild-type C57BL / 6 (WT) primary spleen cells 37℃ group, hTim4 model mouse primary spleen cells 37℃ group, wild-type C57BL / 6 (WT) primary hepatocytes 37℃ group, hTim4 model mouse primary hepatocytes 37℃ group.

[0163] (4) Experimental plan: 10 μl 10 6 TCID 50 / ml of DENV-2Tr1751 strain was incubated with primary spleen cells and primary hepatocytes of wild-type C57BL / 6 and hTim4 model mice at 4°C and 37°C for 2 hours, respectively.

[0164] (5) Sample processing: After 2 hours, cells were collected and RNA was extracted. RT-qPCR was used to detect the RNA content of dengue virus DENV-2.

[0165] 3. Semi-ex vivo proliferation assay

[0166] (1) Virus: 10 6 TCID50 / ml DENV-2Tr1751 strain.

[0167] (2) Primary cells: primary spleen cells and primary hepatocytes of wild-type C57BL / 6 and hTim4 model mice.

[0168] (3) Experimental groups: wild-type C57BL / 6 (WT) primary spleen cell group, hTim4 model mouse primary spleen cell group, wild-type C57BL / 6 (WT) primary hepatocyte group, hTim4 model mouse primary hepatocyte group.

[0169] (4) Experimental plan: 10 μl 10 6 TCID 50 / ml DENV-2Tr1751 strain infected primary spleen cells and primary hepatocytes of wild-type C57BL / 6 and hTim4 model mice.

[0170] (5) Sample processing: Cells were collected at 0, 12, 24 / 36, and 48 hours after infection, and RNA was extracted. RT-qPCR was used to detect the RNA content of dengue virus DENV-2. The detection primer pairs used were: CATGGGTAACTTATGGGAC (SEQ ID NO: 13), GTTTCAGTTCGTGTCTCCA (SEQ ID NO: 14).

[0171] 4. RNA-seq test analysis

[0172] (1) Experimental groups: hTim4 model mouse group and wild-type C57BL / 6 group.

[0173] (3) Sample processing: The total RNA of lung tissue was isolated using the RNeasy Plus Universal Mini Kit (Qiagen, 73404) and the quality was evaluated (OD260 / 280 = 1.8-2.2, OD260 / 230 ≥ 2.0, RIN ≥ 8, 28S:18S ≥ 1.0, > 10 μg). TM The transcriptome-stranded library was prepared by treating the total RNA with the Ribo-Zero magnetic kit (Illumina, 20020596). Initially, ribosomal RNA was removed using the Ribo-Zero magnetic kit (Illumina, 20040526), ​​and the remaining RNA was then fragmented. The first-strand cDNA and double-stranded cDNA were then synthesized and purified. Each blunt fragment of the resulting double-stranded cDNA had an 'A' nucleotide added to its 3' end as a handle for connecting multiple index adapters at both ends of the ds cDNA. Target fragments of 300-420 bp in size were selected and amplified before quantification using a TBS380 mini fluorometer (Tubner Biosystem). Finally, the constructed RNA-seq library was sequenced on the Illumina HiSeq xten / / NovaSeq6000 platform at Novagen (Tianjin). All procedures strictly followed the instructions provided by Illumina.

[0174] (4) Data analysis: The differential expression of genes (DEGs) between wild-type (WT) mice (as a reference group) and hTim4 model mice was analyzed. Subsequently, clean reads were aligned to the mouse reference genome using HIASAT software in directed mode. The expression of each transcript was calculated using the TPM method, and gene abundance was quantified using the RSEM method. DESeq2 / DEGseq / EdgeR with adjusted p-values ​​was used to determine whether the genes showed differential expression. Genes with an adjusted p-value ≤ 0.05 (determined by DESeq2 or EdgeR), as well as a fold change of differentially expressed mRNAs (demRNAs) > 1 or < -1, were considered to be significantly differentially expressed in lung tissues of WT and hTim4 mice.

[0175] In order to clarify the functional characteristics of differentially expressed genes (DEGs), this example also performed Reactome pathway enrichment analysis on the gene list. The top five signaling pathway differential genes were selected for further study. The top ten genes in each pathway were represented by violin plots.

[0176] 5. Cell flow cytometry analysis

[0177] Considering the dependence of infection immunity on T cells and B cells, the present invention further studied the levels of B, T and NK cells using flow cytometry and blood routine tests.

[0178] (1) Experimental groups: hTim4 model mouse group, immunodeficient Rag2 mouse (commercially available, this example uses the model animals in the model animal room of the China National Institute for Food and Drug Control) and wild-type C57BL / 6 group.

[0179] (2) Sample processing: Blood was collected from the medial canthal vein of mice using a test tube containing 1% EDTA (0.5M / L). T, B, and NK cells in peripheral blood were labeled with corresponding antibodies (biolgend), including anti-mouse CD45 antibody, anti-mouse CD19 antibody, and anti-mouse CD49b antibody. Detection and quantification were performed using a BD LSRII flow cytometer and FACSDiva 6.0 software.

[0180] 6. Platelet system testing

[0181] Given the clinical manifestations of bleeding associated with dengue infection, it is important to determine whether hTim4 mice have a normal coagulation system. Therefore, this example also evaluates the coagulation function and vascular fragility caused by the ineffectiveness of hTim4 overexpression by evaluating platelet count, MPV, PDW, PCT, APTT and PT.

[0182] (1) Experimental groups: hTim4 model mouse group and wild-type C57BL / 6 mouse group.

[0183] (2) Sample processing: Blood was collected from the medial canthal vein of mice using a test tube containing EDTA, and routine blood tests were performed using a blood analyzer (Mindary, BC-5000-VET) according to the manufacturer's instructions.

[0184] 7. Statistical analysis

[0185] Statistical analysis was performed using Prism V.8.0 and V.12.0 (GraphPad, La Jolla, California, USA). All data are expressed as mean ± standard deviation (SD). The statistical significance between the two groups was determined by bilateral unpaired Student's t-test, one-way analysis of variance, and two-way analysis of variance. The significance levels are indicated as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: not statistically significant). In the results figures, WT refers to wild-type C57BL / 6 mice, hTim4 refers to hTim4 model mice, DMEM refers to hTim4 model mice intracranial injection of DMEM, and DENV-2 refers to hTim4 model mice intracranial injection of DENV-2.

[0186] 2. Results

[0187] 1. In vitro composite test results

[0188] The results are as follows Figure 7 As shown, the horizontal axis is the dose and the vertical axis is the viral RNA in the complex. The results showed that in in vitro experiments, DENV-2 and hTim4 molecules can directly bind to each other and show dose dependence, suggesting that hTim4 is a receptor molecule for dengue virus.

[0189] 2. Semi-isolated adsorption experiment results

[0190] The results are as follows Figure 8 As shown, Figure 8 A is incubated at 4°C, and B is incubated at 37°C. The results showed that primary spleen cells and hepatocytes of hTim4 model mice could adsorb more DENV-2 viruses at both 4°C and 37°C, and there were statistical differences compared with the control wild-type mouse group.

[0191] 3. Results of semi-in vitro proliferation assay

[0192] The results are as follows Fig. 9 As shown, Fig. 9 A is the situation of primary spleen cells, and B is the situation of primary liver cells. The results show that DENV-2 is more susceptible to infecting primary spleen cells and liver cells of transgenic hTim4 mice, showing higher viral copy numbers.

[0193] The above results verified from the cellular level that hTim4 is the receptor molecule of dengue virus.

[0194] 4. RNA-seq test and cell flow cytometry analysis results

[0195] The results are as follows Fig.10 As shown, A is a volcano map, B is a Reactome functional enrichment analysis map, C is an IFN signaling pathway related gene expression map, and D is a flow cytometry analysis result.

[0196] The results showed that in the transcriptome sequencing results of lung tissues of wild-type (WT) mice and hTim4 model mice, only about 0.6% (185 up and 179 down genes out of 29,253 genes) of hTim4 model mice were expressed differently compared with wild-type mice ( Fig.10 (A).

[0197] Furthermore, the present invention also performed KEGG functional enrichment analysis, and the results are as follows Fig.10 In B, we selected genes related to the IFN signaling pathway for further study. Fig.10 As shown in C, Fig.10 Figure C shows the expression of all IFN signaling pathway-related genes. It was found that the expression of hTim4 molecules had little effect on interferon-related pathways and gene expression profiles, which can be ignored, confirming that the immune function of hTim4 model mice is intact and normal. The results of flow cytometry detection of B, T and NK cell levels are shown in Figure 2. Fig.10 As shown in D, the results showed that there was no significant statistical difference between hTim4 model mice and wild-type C57BL / 6 mice, while Rag2- / - mice showed obvious immune deficiency.

[0198] Compared with the AG129 mouse (a mouse model with double knockout of interferon receptor type 1 and type II) which is currently widely used as a model of dengue infection disease, although this model is susceptible, they have the limitation that the IFN signaling pathway distorts the cytokine response. Dengue virus infection can cause a cytokine storm, which is highly correlated with the IFN signaling pathway. The defective mice cannot respond well to clinical immune responses and are not as good as the hTim4 model mice of the present invention in truly simulating the natural infection immune changes of dengue disease.

[0199] 5. Platelet system test results

[0200] The results are as follows Fig.11 As shown, the results showed that compared with wild-type mice, there were no significant changes in parameters such as platelet count, MPV, PDW, PCT, APTT and PT in hTim4 mice, indicating that hTim4 mice have normal coagulation function and intact blood vessels, that is, hTim4 model mice have normal immune function.

[0201] Example 4

[0202] Dengue hemorrhagic fever evaluation experiment in hTim4 model mice infected with DENV-2.

[0203] 1. Methods

[0204] 1. Experimental groups and attack methods

[0205] (1) Experimental groups: DENV-2 infection group and DMEM control group of hTim4 model mice, and DENV-2 infection group and DMEM control group of wild-type C57BL / 6 mice, denoted as hTim4-DENV-2, hTim4-DMEM, WT-DENV-2, and WT-DMEM, respectively.

[0206] (2) Experimental plan: 30-40 μl 10 3 TCID 50 / ml of DENV-2Tr1751 strain was intracranially injected to infect hTim4 model mice and wild-type C57BL / 6 mice, and equal doses of DMEM were used to infect hTim4 model mice and wild-type C57BL / 6 mice.

[0207] (3) Sample processing: Observe and record the survival rate, body weight and clinical score of each group of mice. When drawing the weight change percentage curve, mice with a weight loss of more than 25% were defined as dead mice according to ethical review requirements. Blood, brain, lung, intestine, spleen, heart, kidney and other tissues of mice were collected.

[0208] 2. Tissue viral load testing

[0209] RT-qPCR was used to detect the content of DENV-2 in different tissues (blood, brain, intestine) at different times (2, 4, 6, 9 dpi for blood, 6 dpi for brain and intestine). The above dpi refers to days post-incubation / inoculation.

[0210] 3. HE staining

[0211] Considering that the symptoms of the brain, lungs, and intestines represent neurological symptoms and systemic symptoms, respectively, which are representative symptoms of dengue hemorrhagic fever, this experiment performed HE staining analysis on the brain, lungs, and intestines.

[0212] Brain, lung, and intestinal tissues of hTim4 model mice and DMEM-infected mice at 10-18 dpi were fixed with formalin, embedded in paraffin, and then transferred to 70% ethanol for preservation. Each tissue sample was placed in a processing box, dehydrated through a continuous alcohol gradient, and finally embedded in a paraffin block. Before immunostaining, 3 μm thick tissue sections were dewaxed with xylene, rehydrated with decreasing concentrations of ethanol, and washed with PBS. Subsequently, H&E staining kit (Leagene; DH0003) was used. A NanoZoomer scanner (HAMAMATSU; C13210-01) was used and previewed using NanoZoomer digital pathology software (NDP.viewer).

[0213] 4. Coagulation time / number of blood drops

[0214] hTim4 model mice were anesthetized with a combination of zoletil 50 and thiazide. Subsequently, the distal part of their tail was resected, and the subsequent hemostatic response was evaluated by quantifying the number of blood drops and measuring the clotting time.

[0215] 5. Prothrombin and activated partial thromboplastin time test

[0216] Blood was collected from anesthetized mice, and sodium citrate was added at a concentration of 10% based on a ratio of 10:1 between the volume of mouse blood and the volume of sodium citrate. 9 mM sodium citrate tube. Anticoagulation was achieved by centrifugation at 3000 rpm for 10-15 minutes, then platelets were removed and the resulting supernatant was collected. The experiment was measured using a prothrombin (PT) test kit (SolarBio, BC8081) and an activated partial thromboplastin time (APTT) test kit (SolarBio, BC8080).

[0217] 6. Neutrophil and white blood cell detection

[0218] Blood was collected from the medial canthal vein of mice using a test tube containing EDTA, and routine blood tests were performed using a blood analyzer (Mindary, BC-5000-VET) according to the manufacturer's instructions.

[0219] 7. HR-LSCI laser speckle blood flow imaging

[0220] After intraperitoneal anesthesia, mice were placed in a prone position and fixed on a mouse holder. After alcohol disinfection, a longitudinal incision was made on the scalp to fully expose the skull. The periosteum was then separated, while the skull was kept moist with saline. Similarly, after alcohol disinfection, a longitudinal incision was made on the abdominal skin to fully expose the mesentery and intestinal tissue. Physiological saline solution was used to maintain moisture during this process. The HR-LSCI laser speckle blood flow imaging system (LSI BFIPLUS, LogiScience Technology Co., Ltd, China) was used to capture mouse blood vessels and blood flow. The parameters were set as specified in the HR-LSCI system setting section below. The system uses a 780nm semiconductor scanning laser as the light source, with an exposure time of 5min, and the reflected light is collected by an sCMOS camera with a recording rate of 1 second / frame and a recording time of 3min. The perfusion information is converted into a two-dimensional perfusion image by the HR-LSCI imaging algorithm and the LSI HMC motion correction algorithm, and then image processing and analysis are performed by the built-in LSI BFI PLUS imaging software.

[0221] 8. Immunofluorescence image-confocal detection

[0222] Intestinal tissue paraffin sections were dewaxed with xylene and continuous alcohol gradients, and then incubated in 3% H2O2 for 15 minutes. Antigens were extracted by boiling with citrate buffer (pH 6.0). After blocking with fetal bovine serum, the following primary antibodies were incubated overnight at 4°C: DENV-2NS1 (ab41616, abcam), CD31 (ab182981, abcam), villin (ab97512, abcam), murinealbumin (ALB, SAB3500217, sigma) and VE-cadherin (ab205336, abcam).). The secondary antibodies Donkey Anti-Rabbit IgG H&L-Alexa Fluor 647 (ab150075) and Goat Anti-Chicken IgY H&L-Alexa Fluor 555 (ab150170, abcam), Goat Anti-Mouse IgG H&L-Alexa Fluor 568 (ab175473, abcam) and Goat Anti-Mouse IgG H&L-Alexa Fluor 594 (ab150160, AbCam) were incubated at 37°C for 1 hour and then detected under a confocal fluorescence microscope (Zeiss LSM900).

[0223] 9. Spatial transcriptome sequencing

[0224] The intestines of hTim4 mice were embedded with SAKURA (4583) and subsequently frozen at -80°C. The embedded tissue blocks were assessed for RNA integrity by Agilent 2100 to ensure RIN values ​​greater than 7. Sequencing libraries were constructed using 10×Genomics' Visium Spatial Gene Expression Slide&Reagent kit (PN-1000184). Each tissue slide was carefully positioned on the capture zone of the Visium gene expression slide and then stained with hematoxylin and eosin (H&E), imaged, and permeabilized. During the permeabilization process, mRNA molecules were released and specifically bound to the oligonucleotides pre-coated on the capture zone. Subsequently, reverse transcription was performed and sequencing libraries were prepared according to the manufacturer's protocol. The resulting raw FASTQ files and pre-existing histological images were processed using Space Ranger (v1.2.0, 10×Genomics) with default parameters. The filtered gene spot matrix and the fiducial aligned low-resolution images were used for downstream data analysis. Gene expression normalization, dimensionality reduction, spot clustering, and differential expression analysis were performed using the Seurat software package.

[0225] Spots with less than 100 genes detected were excluded from further analysis. Normalization between spots was achieved using the built-in SCTransform tool, and then 3000 highly variable genes were selected for principal component analysis (PCA). Spot clustering involved building a graph based on the first 20 principal components (PCs) and then segmenting it at a resolution of 0.5 units. Differential gene expression analysis was performed for each cluster using the Find All Markers function using the nonparametric Wilcoxon rank sum test criterion. Genes with fold changes > 2 and adjusted p-values ​​< 0.05 were considered significantly differentially expressed genes (DEGs). Enrichment testing of candidate gene sets was performed using the cluster Profiler R package based on the principle of hypergeometric distribution. Pathways with adjusted p-values ​​below 0.05 were considered significantly enriched, and reactome enrichment analysis was used to analyze DEG functions.

[0226] 10. Cytokine detection

[0227] The collected blood samples of hTim4 model mice and DENV-2 infected patients were centrifuged at 10000rpm to obtain serum. The cytokine concentration in serum was quantified by MSD (Meso Scale Discovery) method using Bio-Plex 200 system and Bio-Plex Pro mouse cytokine 23-plex Assay (M60009RDPD, Bio-Rad, USA) and Bio-Plex Pro human cytokine 27-plex Assay (#M500KCAF0Y, Bio-Rad, USA) according to the instructions (10014905, Bio-Rad, CA, USA).

[0228] 11. Statistical analysis

[0229] Statistical analysis was performed using Prism V.8.0 and V.12.0 (GraphPad, La Jolla, California, USA). All data are presented as mean ± standard deviation (SD). Two-sided unpaired Student's t-test, one-way ANOVA, and two-way ANOVA were used to determine the statistical significance between the two groups. The significance levels are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not statistically significant). In the result figures, WT refers to wild-type C57BL / 6 mice, hTim4 refers to hTim4 model mice, DENV-2 refers to DENV-2 infection, and DMEM refers to the control injected with DMEM, such as: WT-DENV-2 refers to wild-type C57BL / 6 mice infected with DENV-2, hTim4-DENV-2 refers to hTim4 model mice infected with DENV-2, WT-DMEM refers to the control injected with DMEM of wild-type C57BL / 6 mice, and hTim4-DMEM refers to the control injected with DMEM of hTim4 model mice.

[0230] 2. Results

[0231] Currently, there is no in vivo evidence of potential dengue receptor-mediated virus infection. Although dengue hemorrhagic fever is common in dengue patients, it is difficult to replicate in animal models. However, the experimental results of the present invention show that hTim4 model mice are highly sensitive to DENV-2 infection and can highly simulate the clinical manifestations of dengue hemorrhagic fever.

[0232] 1. Experimental results on the sensitivity of hTim4 model mice to DENV-2 infection.

[0233] Fig.12The viremia graphs of hTim4 model mice infected with DENV-2 (hTim4-DENV-2) and wild-type C57BL / 6 mice infected with DENV-2 (WT-DENV-2) are shown in the figure. The test data was standardized in the figure, and the ordinate is the relative multiple relationship between hTim4-DENV-2 and WT-DENV-2. The results showed that hTim4 model mice developed viremia from the second to the ninth day after infection, and reached a peak on the fourth day.

[0234] Fig.13 The statistical chart of mouse survival period shows that DENV-2 (10 3 TCID 50 / ml) infected 6-week-old hTim4 model mice (hTim4-DENV-2), the mortality rate was 50% on the 12th day after infection (dpi), and all mice died at 18dpi. In contrast, at least 25% of WT mice (WT-DENV-2) survived to the end of the experiment. There was no death in WT-DMEM and hTim4-DMEM that were not infected with DENV-2.

[0235] Fig.14 This is a statistical chart of mouse weight changes. It can be seen that starting from the 6th day after infection, the weight of hTim4 model mice (hTim4-DENV-2) decreased significantly, exceeding 25%, meeting the death criteria.

[0236] Fig.15 This is a scoring chart for clinical symptoms of mice. The scoring criteria are ruffled hair (0-5), hunched back (0-5), slow movement (0-5), tremor (0-5), forelimb paralysis (0-5), hindlimb paralysis (0-5), weight loss (0-5), dying and death (0 / 5). The specific severity of each symptom is scored from 0 to 5 points from mild to severe. Finally, the scores of each symptom are added together to get the clinical score. It can be seen that starting from 8 dpi, the clinical symptom scores of hTim4 model mice infected with DENV-2 (hTim4-DENV-2) are higher than those of WT mice (WT-DENV-2), indicating that the course of the disease is more severe.

[0237] Fig.16 The figure shows the viral load of multiple organ tissues in mice. The test data were standardized. The ordinate is the relative multiple relationship between hTim4-DENV-2 and WT-DENV-2. A is the relative viral load in the brain, and B is the relative viral load in the intestine. The results can further confirm that at 6 dpi, the viral load in the brain and intestinal tissues was high (10 6 ～7 and 10 2～3 copies / g), but was not detected in lung tissue.

[0238] These results provide the first in vivo evidence supporting the role of the hTim4 receptor in mediating viral entry and demonstrate that hTim4 model mice are highly susceptible to DENV-2 infection.

[0239] 2. Evaluation of dengue hemorrhagic fever symptoms.

[0240] Systemic bleeding is the main symptom of severe DHF patients, which is difficult to replicate in animal models. Therefore, the present invention studied the multi-organ bleeding characteristics of DENV-2 infected hTim4 mice. The results showed that hTim4 model mice faithfully replicated the clinical manifestations of dengue hemorrhagic fever (DHF).

[0241] Fig.17 This is a picture of multi-organ bleeding in mice. The results show that obvious signs of bleeding were observed in the brain, lungs, intestinal tissue, heart, liver and spleen of hTim4 model mice infected with DENV-2. Of particular note is that obvious bleeding spots were observed in the intestines.

[0242] Fig.18 This is the HE staining image of the brain pathology of hTim4 model mice. Pathological analysis shows that bleeding in hTim4 model mice infected with DENV-2 mainly occurs in the cerebral cortex (+), accompanied by neuronal necrosis (→) and perivascular infiltration (*), which is associated with paralysis and ataxia symptoms and bleeding.

[0243] Fig.19 HE staining of hTim4 model mouse lung pathology, lung sections show obvious pulmonary hemorrhage (+), hemosiderin deposition (▲) and alveolar edema (★), which is consistent with the dyspnea phenotype and red lung appearance of the mouse. It is worth noting that, unlike other microbial infections such as SARS-CoV-228, no large number of immune cell infiltration or aggregation was observed in brain or lung sections, indicating that dengue infection has unique pathological characteristics or pathogenesis.

[0244] Fig. 20 This is the HE staining image of intestinal pathology of hTim4 model mice. Pathological examination showed that compared with the uninfected control group, the intestinal villi of hTim4 model mice were severely damaged and bleeding after DENV-2 infection, manifested by a large number of blood cells, irregular cell nuclei, and atrophic cytoplasm.

[0245] Fig.21 This is a schematic diagram of the coagulation situation of hTim4 model mice, where A and B are the coagulation time and blood drop number diagrams, respectively. The results show that compared with the uninfected control group, the coagulation time of hTim4 model mice infected with DENV-2 began to change 4 days after infection and then significantly prolonged; the blood drop number increased significantly 8 days after infection (P<0.05).

[0246] Fig. 22 Schematic diagram of prothrombin time and partial prothrombin activation time in hTim4 model mice, where A is the mean corpuscular volume (MCV), B is the platelet count, C is the activated partial thromboplastin time, D is the prothrombin time, lane 1 in BD is the DMEM control without virus infection, lane 2 is the result at 3dpi, and lane 3 is the time of death (succumed). It was observed that compared with the uninfected control group, the mean corpuscular volume (MCV) of hTim4 model mice infected with DENV-2 decreased (P<0.05), which is consistent with the manifestation of dengue hemorrhagic fever. In addition, the platelet count (PLT) is also similar to the clinical thrombocytopenia cases observed during DENV-2 infection. The APTT and PT times of the dead mice were prolonged, indicating impaired coagulation function.

[0247] Fig.23 The figure shows the neutrophils and white blood cells of hTim4 model mice, where A represents white blood cells and B represents neutrophils. The results show that compared with the uninfected control group, the number of white blood cells and neutrophils in hTim4 model mice was significantly reduced after DENV-2 infection.

[0248] The above comprehensive evaluation of coagulation time, PLT count, activated partial thromboplastin time (APTT), prothrombin time (PT), and recording of tail tip drop counts were used to comprehensively evaluate the experimental results of coagulation system damage caused by DENV-2 infection, confirming the occurrence of coagulation dysfunction caused by DENV-2 infection in hTim4 model mice. The observed systemic bleeding characteristics indicate that hTim4 mice faithfully replicate the clinical manifestations of dengue hemorrhagic fever (DHF), especially intestinal bleeding.

[0249] 3. Study on intestinal bleeding and failure caused by DENV-2 infection.

[0250] In severe clinical cases, the occurrence of massive gastrointestinal bleeding can lead to hemorrhagic shock (DSS), organ failure, and ultimately death. Therefore, the following studies focus on intestinal bleeding and failure caused by DENV-2 infection. Capillary leakage, characterized by changes in blood flow velocity and vascular permeability, has been reported as a typical feature of DHF bleeding. The present invention uses the HR-LSCI laser speckle blood flow imaging system to evaluate blood flow rate and perfusion pressure, where stronger red image signals indicate more adequate and faster blood flow.

[0251] In this example, hTim4 model mice infected with DENV-2 in the hTim4-DENV-2 group were divided into mild, moderate and severe groups according to their intestinal changes, specifically based on pathological scores and perfusion pressure changes. The specific groups are as follows:

[0252] Mild cases: pathological score (0-1) or perfusion pressure (0-10%);

[0253] Moderate: pathological score (1-2) or perfusion pressure (10-30%);

[0254] Severe cases: pathological score (2-3) or perfusion pressure (more than 30%).

[0255] The above percentages of perfusion pressure refer to the percentage change of perfusion pressure compared with the healthy control group.

[0256] The results showed that there were no mild cases, and the patients were subsequently analyzed using healthy, moderate and severe control groups.

[0257] Fig.24 The figure is a schematic diagram of intestinal tissue bleeding, congestion, and even intestinal failure in hTim4 model mice, where A is a photo of the intestine of each group of hTim4 model mice under a bright light microscope, B is an image of the intestinal blood flow of each group of hTim4 model mice, C is the intestinal perfusion pressure of each group of hTim4 model mice, and D is a graph of the intestinal systolic and diastolic pressure of each group of hTim4 model mice. The results show that in the healthy group (DMEM, health), a vibrant and healthy intestine was observed under a bright light microscope, with abundant blood flow in both systolic and diastolic phases. However, in the severe group (severe), the ischemic and gangrenous intestine appeared black, as confirmed by its blood flow image, with only a weak red signal detected. The blood flow of the moderate group was between the healthy group (DMEM, health) and the severe group (severe), indicating that the higher the severity of DHF, the slower the blood flow.

[0258] In addition, significant intestinal bleeding may lead to decreased perfusion pressure, therefore, the present invention further analyzed the perfusion pressure of the three groups of model mice. Our analysis showed that compared with the healthy group, the perfusion pressure of the moderate group decreased by >28.75%, and the perfusion pressure of the severe group decreased by >42.26%, showing a linear downward trend. Based on this, we assume that the occurrence of DSS in the severe group is due to a substantial decrease in intestinal perfusion pressure (>40%). Overall, these findings confirm the lack of blood supply in the intestines of mice infected with hTim4.

[0259] Furthermore, the present invention attempts to investigate the causes and consequences of intestinal bleeding by using immunofluorescence staining of the villus marker villin. Fig.25It is the immunofluorescence staining diagram of hTim4 model mice, and it can be seen that the cell nucleus is arranged irregularly (blue), the cytoplasm is atrophic, and the villus layer is atrophic (purple). In order to evaluate the permeability of the capillary wall, this embodiment uses albumin (ALB, yellow) and CD31 (purple) for joint staining, wherein ALB is usually confined to the blood vessel, but can penetrate the blood vessel wall, which is due to the plasma leakage in the tissue infected by DENV-2, resulting in changes in vascular permeability. The presence of DENV-2 virus (brown) and its receptor hTim4 (green) co-localization further supports the hypothesis of the present invention, that these pathological features are induced by DENV-2 infection. In addition, the present invention also uses immunostaining analysis to evaluate the expression of VE-cadherin protein (purple, purple) in DENV-2 positive tissues. However, compared with the DMEM control group, no obvious changes were observed, which indicates that ve-cadherin may not be the main factor causing vascular leakage.

[0260] In summary, hTim4 mice showed characteristics of dengue hemorrhagic fever, as well as vascular leakage and intestinal failure; ischemia and bleeding jointly exacerbated the occurrence of intestinal failure.

[0261] 4. Spatial transcriptomics results

[0262] The genes that cause intestinal ischemia and bleeding symptoms and their spatial distribution remain poorly understood. To investigate the potential correlation between gene expression and increased vascular permeability in the intestine, spatial transcriptomics was used in this study. Seruat software was used to perform normalization, dimensionality reduction, spot clustering, cell type identification, and differentially expressed gene (DEGs) analysis on all genes obtained by spatial transcriptome sequencing.

[0263] Fig.26 These are the spatial transcriptome sequencing maps of the dengue virus DENV-2 infected group and the DMEM uninfected group of hTim4 model mice, where A is the HE staining map and three-dimensional tissue localization map of the DENV-2 infected group and the DMEM uninfected group, and B is the two-dimensional coordinate map of the DENV-2 infected group and the DMEM uninfected group, showing the spatial distribution of gene points in the DMEM group and the DENV-2 group.

[0264] Fig. 27The figure is a schematic diagram of the comparison of cell types in the intestinal tissues of the dengue virus DENV-2 infected group and the uninfected group, where A is a schematic diagram of the uninfected DMEM group, B is a schematic diagram of the DENV-2 infected group, and C is a tissue cell type localization distribution map, where different colors represent different cell types. A total of 14 cell types were detected: tuft cells, B cells, T cells, smooth muscle cells, paneth cells, NK cells, mesenchymal cells, macrophages, immune cells, goblet cells, epithelial cells, endothelial cells, and blood endothelial cells. The results showed that there were significant differences in the spatial distribution of intestinal tissue cell types between the dengue virus DENV-2 infected and uninfected groups. NK cells, paneth cells, tuft cells, T cells, macrophages, and B cells showed co-localization in the DENV-2 group, and their numbers increased specifically, indicating that there was an immune response after DENV-2 infection. Given that NK, T, B, and macrophages are known to secrete inflammatory regulators, the increase in immune cell populations may lead to the occurrence of cytokine storms.

[0265] Fig.28 This is the differential gene analysis between the dengue virus DENV-2 infection group and the DMEM uninfected group in the hTim4 model mouse, where A is the cell cluster diagram of the DENV-2 infection group and the DMEM uninfected group, B is the Reactome functional analysis heat map of the differential genes in each cluster, and C is the violin map of the top 10 genes of the signaling pathway marked by * in Figure B. According to the expression level, all cells from the DMEM and DENV-2 infection groups can be divided into 9 clusters, ranging from 0 to 8 (A).

[0266] The present invention further analyzed the expression and number of differentially expressed genes (DEGs) in the nine clusters, and in order to determine their functions, Reactome functional enrichment analysis was performed. The results showed that DEGs were mainly enriched in pathways related to coagulation dysfunction, cell connectivity, and immunity (B). Since cluster 0 had the largest number of DEGs and the most representative functional analysis, the top five signaling pathways in cluster 0, including platelet activation / aggregation, hemostasis, extracellular matrix organization, innate immune system, and neutrophil degranulation, were selected for further study. Interestingly, it was found that most of the DEGs in cluster 0 were downregulated, Fig.10Center C shows the 10 most significantly downregulated genes involved in each signaling pathway, col1a2, col1a1, clu, ceacm1, col3a1, klk1, reg3g, reg3b, hbb-bs, and neu1. These 10 genes have been reported to be involved in coagulation / bleeding responses, while several others regulate cytokines, such as klk1 regulating IL-6 activity and reg3g inhibiting IL-6 / IL-8 secretion, suggesting that they are related to cytokine secretion or activity. However, fewer genes with roles in bleeding, ischemia, or even increased vascular permeability are available. In summary, using spatial transcriptomics data, molecular signatures of intestinal bleeding and ischemia in hTim4-infected mice showed increased immune cells and downregulated genes.

[0267] Since the functions of the 10 significantly down-regulated genes were related to cytokine secretion, we performed protein-protein interaction (PPI) network analysis to evaluate their interactions.

[0268] Fig.29 This is the interaction diagram of differentially expressed genes and cytokines between the hTim4 model mouse DENV-2 infected group and the uninfected group. The results showed that the 10 most downregulated genes in each pathway, including platelet activation and aggregation, hemostasis, extracellular matrix organization, innate immune system and neutrophil degranulation, were involved in cytokine secretion and inflammatory mediators. The details are shown in the following table.

[0269] Table 7. Top 10 most downregulated genes in each pathway

[0270]

[0271] Subsequently, we selected col1a1, col1a2, ceacam1, clu, klk1, col3a1, reg3b, reg 3g, hbb-bs, and neu1 as representative genes in each pathway ( Fig.28 RT-qPCR validation was performed (red box in center C).

[0272] Fig.30 The expression levels of differentially expressed genes in the dengue virus DENV-2 infected and uninfected groups of hTim4 model mice. The results showed an expression pattern consistent with the spatial transcriptome data.

[0273] We then used flow cytometry to assess the Th1 and Th2 cytokine bias in the sera of hTim4 mice following DENV-2 infection. Fig.31The following is a graph showing changes in cytokines in hTim4 model mice. The results showed that the levels of IL-6 (P<0.01) and IL-10 (P<0.05) secreted by Th2 cells were increased, and the expression of inflammatory mediators MCP-1 (P<0.05), MIP-1β (P<0.05), and RANTES (P<0.05) that promote Th2 differentiation were also increased. In contrast, the levels of IL-2, IL-12 (p40 and p70), and IFN-γ, which are classified as Th1-type cytokines, changed little. These findings strongly suggest that after DENV-2 infection, hTim4 mice developed a systemic cytokine storm characterized by a prominent Th2-type bias.

[0274] Example 5

[0275] To verify the bias of systemic cytokines characterized by a prominent Th2-type bias found in Example 4 in humans, this example further analyzed sera obtained from DF or DHF patients and healthy individuals during the DENV-2 epidemic in southern China from 2014 to 2015 (the detection process was reviewed by the Ethics Committee of the Yunnan Institute of Parasitic Diseases (No. 03)). The results are as follows Fig.32 As shown, Fig.32 The results of human cytokine changes are very consistent with our results in hTim4 mice. These analyses revealed that DENV-2 infection induced a significant cytokine storm in human subjects and also showed a clear Th2-type bias.

[0276] In summary, both humans and hTim4 mice exhibited a Th2-type cytokine storm rather than a Th1-type response upon DENV-2 infection. The high consistency between these findings emphasizes the reliability and fidelity of the hTim4 model mouse as an experimental model for replicating the immune response triggered by DENV infection.

Claims

1. A method for establishing an animal model of dengue hemorrhagic fever expressing hTim4, It is characterized in that The following steps are involved: The vector containing the DNA fragment of human Tim4 is transferred into the mouse fertilized egg, the mouse is transgenicized with the Tol2 transgenic system, and the surviving fertilized egg is implanted into the pseudo-pregnant recipient mouse. After screening, positive mice with high expression of hTim4 are obtained, and the dengue virus is injected intracranially into the positive mouse.

2. The method for establishing a dengue hemorrhagic fever animal model expressing hTim4 as claimed in claim 1, It is characterized in that The following steps are involved: Targeting vector construction: Construction of a targeting vector containing a DNA fragment of human Tim4; Cultivate F0 generation mice: inject the positive targeting vector and the vector encoding Tol2 enzyme into the fertilized egg cells of mice, and transplant the surviving fertilized eggs into pseudo-pregnant mice, and select the positive mice among the born mice, which are the F0 generation mice for future use; Breeding: The F0 generation mice were mated with wild-type mice of the same strain, and the mice expressing hTim4 were obtained as positive mice; Infection: Inject dengue virus into the positive mice intracranially to obtain the dengue hemorrhagic fever animal model.

3. The method for establishing a dengue hemorrhagic fever animal model expressing hTim4 as claimed in claim 2, It is characterized in that The targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-WPRE. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-WPRE-PolyA. Alternatively, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP, further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP-WPRE, and further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP-WPRE-PolyA.

4. The method for establishing a dengue hemorrhagic fever animal model expressing hTim4 as claimed in claim 3, It is characterized in that The DNA sequence of hTim4 is shown in SEQ ID NO: 1; And / or, the sequence of CAG is shown in SEQ ID NO: 2; And / or, the sequence of KOZAK is gccacc; And / or, the sequence of P2A is shown in SEQ ID NO:3; And / or, the sequence of EGFP is shown in SEQ ID NO:4; And / or, the sequence of the WPRE is shown in SEQ ID NO:5; And / or, the sequence of PolyA is shown in SEQ ID NO:6; And / or, the targeting vector sequence is shown in SEQ ID NO:

7.

5. The method for establishing a dengue hemorrhagic fever animal model expressing hTim4 as claimed in claim 1, It is characterized in that The dengue virus is dengue virus type 2; And / or, the vector is a Tol2 vector; And / or, the mouse is a C57BL / 6 mouse.

6. The method for establishing a dengue hemorrhagic fever animal model expressing hTim4 as claimed in claim 2, It is characterized in that The Tol2 sequence encoding the Tol2 enzyme is shown in SEQ ID NO:8; And / or, the vector sequence encoding the Tol2 enzyme is shown as SEQ ID NO:

15.

7. The method for establishing a dengue hemorrhagic fever animal model expressing hTim4 as claimed in claim 2, It is characterized in that The positive mice were screened using the primer pair shown in SEQ ID NO:9-SEQ ID NO:10; And / or, the targeting vector contains an EGFP sequence fragment, and positive mice are screened by in vivo fluorescence detection using a CT imaging system or by using a primer pair as shown in SEQ ID NO:11-SEQ ID NO:

12.

8. A method for establishing a dengue hemorrhagic fever susceptible animal model expressing hTim4, It is characterized in that The method comprises the following steps: transferring a vector containing a DNA fragment of human Tim4 into a mouse fertilized egg, subjecting the mouse to transgenic treatment with a Tol2 transgenic system, and implanting the surviving fertilized egg into a pseudo-pregnant recipient mouse, and obtaining positive mice with high expression of hTim4 through screening.

9. The method for establishing a dengue hemorrhagic fever susceptible animal model expressing hTim4 according to claim 8, It is characterized in that The following steps are involved: Targeting vector construction: Construction of a targeting vector containing a DNA fragment of human Tim4; Cultivate F0 generation mice: Inject the positive targeting vector and Tol2 mRNA into mouse fertilized egg cells, and transplant the surviving fertilized eggs into pseudo-pregnant mice. Screen the positive mice among the born pups, which are the F0 generation mice for future use; Breeding: The F0 generation mice are mated with wild-type mice of the same strain, and the mice expressing hTim4 are obtained as positive mice.

10. The method for establishing a dengue hemorrhagic fever susceptible animal model expressing hTim4 according to claim 8, It is characterized in that The targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-WPRE. Further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-WPRE-PolyA. Alternatively, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP, further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP-WPRE, and further, the targeting vector contains a sequence fragment of CAG-KOZAK-hTim4-P2A-EGFP-WPRE-PolyA.

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