Mouse model infected with coxsackie virus A16 and construction method thereof

Abstract

The invention discloses a mouse model infected with coxsackie virus A16 and a construction method of the mouse model, and the construction method comprises the following specific steps: connecting a left homologous arm LHA of a Rosa26 site to a plasmid pCAG-GFP-N1, and successfully constructing the plasmid pCAG-GFP-N1-Rosa26 LHA. Then carrying out PCR (Polymerase Chain Reaction) amplification on the Rosa26-RHA fragment, the hSCARB2 fragment and the wpre-polyA fragment by using a high-fidelity enzyme; the preparation method comprises the following steps: carrying out enzyme digestion by using Hind3, recovering a large fragment as a connecting vector for later use, and successfully constructing the three fragments onto the vector by using a Gibson Assembly one-step method, thereby obtaining the required target vector pmRosa26-CAG-hSCARB2. Then mixing the target vector with targeted sgRNA and Cas9 mRNA, co-injecting the mixture into a fertilized egg of a C57 mouse, then transplanting the fertilized egg into a fallopian tube of a pseudopregnant ICR female mouse, and finally obtaining an hSCARB2 fixed point (Rosa26) knock-in mouse. Through intracranial injection of the CVA-16 virus, the result proves that the knock-in mouse can be successfully infected with the CVA-16 virus at the age of 10 days, 21 days and 30 days, and the model has serious phenomena such as posterior limb paralysis and death for the first time, so that a powerful tool is provided for vaccine evaluation and drug screening.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a mouse model infected with Coxsackievirus A16 and a construction method thereof. Background Art

[0002] Hand, foot, and mouth disease (HFMD) is an infectious disease caused by human enterovirus. Fecal-oral transmission and contact are considered to be the main transmission routes of HFMD, which is related to Coxsackievirus A16 (CVA-16) infection. Most HFMD outbreaks worldwide since 2017 have been caused by CVA-16. Currently, there is no specific antiviral drug for HFMD. The current vaccines for HFMD include enterovirus 71 (EV-71), which can effectively prevent EV-71-related HFMD, but have no cross-protective effect against HFMD caused by CVA-16, CVA-10, and other enteroviruses. Based on the global population mobility and my country's specific social background and population composition, it is very important to consider the development of a CVA-16 vaccine.

[0003] The mouse model that can successfully infect CVA-16 reported so far is similar to the mouse model of EV-71 infection, and can only infect suckling mice less than 7 days old. The young age of infection and the short infection period seriously limit the role of mice in basic research and vaccine drug evaluation. At the same time, the small size of suckling mice also brings unavoidable difficulties in operation. Secondly, the suckling mouse model cannot fully reflect the whole process of infection and pathological effects. Although some researchers have used mice adapted to virus strains or immunodeficient newborn mice to establish CVA-16 animal models, however, the mutations of the adapted virus strains, the entry pathways of the virus, and mice with immune defects cannot accurately reflect the pathogenesis of human diseases. Therefore, it is urgent to construct new experimental animal models to overcome these limitations. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for constructing a Coxsackievirus A16 mouse model, which comprises the following steps: 1) Using the plasmid pCAG-GFP-N1 as the basic skeleton, the left homology arm LHA of the Rosa26 site was connected to pCAG-GFP-N1 to construct the plasmid pCAG-GFP-N1-Rosa26 LHA; 2) Then, the Rosa26-RHA and hSCARB2 fragments and the wpre-polyA fragment were amplified by PCR, and after being digested with Hind3, the large fragments were recovered and used as connection vectors for later use; 3) The large fragment in 2) was successfully constructed into the pCAG-GFP-N1-Rosa26 LHA in 1) using the Gibson Assembly one-step method to obtain the plasmid pmRosa26-CAG-hSCARB2 Targeting Vector; that is, the donor sequence of the target fragment; 4) When the fertilized egg has obvious pronuclei, dilute it with TE buffer, and co-inject Cas9 mRNA, sgRNA targeting hSCARB2, and the donor sequence of the target fragment obtained in 3) into the fertilized egg; after the injection, move the fertilized egg into another fresh M2 culture medium for culture; 5) After in vitro culture, the cells were transplanted into the oviduct of pseudo-pregnant ICR female mice and raised for delivery; after 20-21 days, F0 generation mice were obtained, and F0 generation positive mice were mated with wild-type mice to obtain FI generation positive mice, that is, hSCARB2-KI mice; 6) CV-A16 was then injected intracranially into the hSCARB2-KI mice, thereby successfully constructing a mouse model infected with Coxsackievirus A16.

[0005] Furthermore, in 4), the final concentration of the sgRNA targeting hSCARB2 is 12.5 ng / μl; the final concentration of the Cas9 mRNA is 25 ng / μl; and the final concentration of the donor sequence of the target fragment is 10 ng / μl.

[0006] Furthermore, the in vitro culture time in 5) is 2-3 hours.

[0007] Furthermore, the construction method also includes: 7) genotyping the Coxsackievirus A16-infected mouse model constructed in 6) by PCR and WB, detecting the viral load and pathological histological changes by RT-qPCR and HE staining, and the results show that the Coxsackievirus A16-infected mouse model was successfully constructed. Beneficial Effects

[0008] 1. hSCARB2 is targeted and inserted into the Rosa26 site. It will not cause a strong immune rejection reaction and be eliminated in mice, and can be expressed for a long time. Because Rosa26 was first discovered by Friedrich and Soriano when studying gene mutations in mouse embryonic stem cells. Located on chromosome 6 of the mouse, this region is very easy to insert genes. No functional expression protein has been found so far. It is easy to perform homologous recombination. The protein knocked into this region can be widely expressed in all cell types and developmental stages. It will not affect the expression and function of other endogenous genes. Therefore, the Rosa26 site of mice is often used as a "safe haven" for gene targeting; therefore, the present invention also achieves stable systemic expression of exogenous genes. It avoids phenotypic abnormalities that may be caused by silencing endogenous gene expression. This is also one of the obvious differences between the present invention and the existing hSCARB2 KI mice constructed by ESCs.

[0009] 2. As a targeted gene knock-in strategy, the number of mice required is less than that of BAC transgenic and PiggyBac transgenic, which greatly improves work efficiency and reduces production costs.

[0010] 3. The hSCARB2 KI mouse uses the relatively safe CRISPR-Cas9 technology, with a broad CAG as the promoter, adding the wpre element and bGH-polyA sequence. The vector using the wpre element can enhance the expression of the target gene, which has a great advantage in improving the efficiency of protein expression. Compared with the stronger signals of SV40 late polyA and rbGlobpolyA (which are considered to terminate transcription more effectively), bGH with a slightly weaker poly(A) signal can enhance the stability of mRNA and provide a longer template for the continuous synthesis of proteins. Secondly, the bGH PolyA sequence can promote the nuclear transport of mRNA, making it easier to enter the cytoplasm and translate. Therefore, the combination of wpre elements and bGH-polyA designed in this study can improve the stability and translation efficiency of hSCARB2. This targeting strategy also enables hSCARB2 to be widely expressed in mouse tissues, giving it a significant advantage in reflecting the damage of viruses to different tissues and organs, which reflects its innovation in terms of vector structure design. In addition, there is little or no off-target damage in the first animals (F0 generation); more importantly, the CRISPR-Cas9 system is extremely easy to use due to its rapidity and ease of operation. Any laboratory can carry out the work, making its promotion and application more convenient and faster.

[0011] 4. The CVA-16 suckling mouse model has problems such as differences from human receptors and can only indirectly evaluate the protective effect of vaccines, which brings certain difficulties to the development and application of vaccines. However, this model has achieved the insertion of CVA-16 human receptors (hSCARB2) and can successfully infect CVA-16 virus at 10 days, 21 days, and 30 days of age. In addition, 10-day-old mice developed serious phenomena such as hind limb paralysis and death after virus infection. Further research characteristics showed that the expression of hSCARB2 in brain tissue and hind limb tissue of heterozygous mice reached a peak, and after infection with CVA-16, high expression of CVA-16 antigen was detected in brain tissue and hind limb tissue, which proved for the first time that the expression of viral antigens and viral receptors in this KI mouse after intracranial virus infection was identical, and both had a tendency to brain and hind limb tissues.

[0012] 5. This model provides a powerful tool for the evaluation of CVA-16 vaccines and related drug screening, and also has extremely high application value in studying the mechanism of CVA-16 infection and nerve damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above and other objects, features and advantages of the present application will become more apparent by describing in more detail the exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0014] Figure 1 Schematic diagram of the targeting vector of the embodiment of the present application comprising 5 elements; in the figure, Rosa26-LHA: refers to the left homology arm of the Rosa26 gene site; CAG promoter: refers to the CAG promoter; hSCARB2: refers to scavenger receptor 2; wpre-polyA: refers to the woodchuck element (commonly used when constructing a vector) followed by a polyA tail to improve the translation efficiency of mRNA; Rosa26-RHA: refers to the right homology arm of the Rosa26 gene site.

[0015] Figure 2 This is the gel electrophoresis diagram of the intermediate vector plasmid pCAG-GFP-N1-Rosa26 LHA identified by PCR.

[0016] Figure 3 This is the sequencing comparison result of the intermediate vector plasmid 4# clone.

[0017] Figure 4 This is the complete plasmid map of pmRosa26-CAG-hSCARB2 Targeting Vector.

[0018] Figure 5This is the gel electrophoresis diagram of colony identification of plasmid pmRosa26-CAG-hSCARB2 targeting Vector.

[0019] Figure 6 This is the sequencing comparison result of 12# clone bacteria.

[0020] Figure 7 This is the electrophoresis diagram of 7 KI mice carrying the hSCARB2 gene identified by PCR.

[0021] Figure 8 This is the sequencing result of the partial sequence (723bp) of hSCARB2 KI mouse.

[0022] Fig. 9 This is a diagram showing the expression of hSCARB2 protein identified by Western blot.

[0023] Fig.10 These are the viral load graphs detected by RT-gPCR in brain, heart, liver, lung and hind limb tissues after virus challenge; A, the viral load graphs detected by RT-qPCR in brain, heart, liver, lung and hind limb tissues of 10-day-old mice after virus challenge; B, the viral load graphs detected by RT-GPCR in brain, heart, liver, lung and hind limb tissues of 21-day-old mice after virus challenge; C, the viral load graphs detected by RT-gPCR in brain, heart and hind limb tissues of 30-day-old mice after virus challenge.

[0024] Fig.11 These are the pathological results of brain and hind limb tissues after infection (HE staining). DETAILED DESCRIPTION

[0025] The embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0026] Traditional methods, such as using gene targeting to produce mutant mice, require many steps, including constructing targeting vectors, establishing embryonic stem cells (ESCs) clones with the desired mutation, generating chimeric mice, and confirming germline transmission of the desired mutation. It may take more than a year to obtain mutant mice; in addition, the process of obtaining ESCs is complicated and costly, and usually needs to be carried out under a strictly controlled environment, which limits the popular use of laboratories; secondly, ESCs genomic instability is one of the reasons for the failure of germline transmission, compared with the genomic instability seen in cultured ESCs cells, therefore, this application uses CRISPR-Cas9 technology for targeting.

[0027] Example 1: Construction of pmRosa26-CAG-hSCARB2 targeting vector

[0028] Main reagents: KOD OneTM PCR Master Mix -Blue (Toyobo Co., Ltd., Japan); 2×Es TaqMasterMix (Dye) (Beijing Kangwei Century); DNA Ligation Kit Ver.2.1 (takara); MinElute PCR Purification Kit (50) (QIAGEN); EndoFree Plasmid Maxi Kit (QIAGEN); pEASY®-BasicSeamless Cloning and Assembly Kit (Beijing Kangwei Century); restriction endonucleases SpeI (R3133V) and HindIII (R3104V) (NEB, USA).

[0029] Main experimental instruments: PCR instrument (BIO RAD, USA); dry thermostat (Hangzhou Miou Instrument Co., Ltd.); electrophoresis instrument (BIO RAD, USA), constant temperature oscillator (BIO RAD, USA).

[0030] The targeting vector in this experiment contains 5 elements, such as Figure 1 As shown. We constructed the plasmid pCAG-GFP-N1 as the basic skeleton. First, the left homology arm LHA of the Rosa26 site was connected to the plasmid pCAG-GFP-N1, and the plasmid pCAG-GFP-N1-Rosa26 LHA was successfully constructed. Then, the Rosa26-RHA and hSCARB2 fragments and the wpre-polyA fragment were amplified by PCR using a high-fidelity enzyme and recovered for later use; Hind3 was used to cut pCAG-GFP-N1-Rosa26 LHA and the large fragment was recovered as a connection vector for later use, and then the three fragments were successfully constructed into the vector using the Gibson Assembly one-step method, that is, the pmRosa26-CAG-hSCARB2 Targeting Vector plasmid was obtained.

[0031] 1. Construction of the intermediate vector pCAG-GFP-N1-Rosa26 LHA The vector pCAG-GFP-N1 (constructed by Beijing Weitongda Biotechnology Co., Ltd.) was digested with restriction endonuclease Spe1, and the 5.5 kb fragment was recovered for use. The fragment Rosa26-LHA to be connected was obtained by PCR amplification using KOD OneTM PCR Master Mix-Blue-, and the amplification primers were rosa-LHA-spe1F and rosa-LHA-spe1R (synthesized by Beijing Deaoping Biological Co., Ltd.). The primer sequences are shown in Table 1. The amplification template was the plasmid mROSA FOXO1-7MU targeted allele (constructed by Weitongda Biotechnology Co., Ltd.). Then the vector pCAG-GFP-N1 (Spe1) and the fragment Rosa26-LHA were connected using the DNA Ligation KitVer.2.1 kit. After transformation, single clones were selected and PCR identification was performed using primers Rosa-LHA-jcF2 and rosa-LHA-jcR2. The size of the positive band was 864 bp, and the identification gel electrophoresis diagram was shown in Figure 2 . The 4# bacterial solution was selected for sequencing. The sequencing results were found to be completely correct after comparison. The intermediate vector pCAG-GFP-N1-Rosa26 LHA was successfully obtained. The sequencing results are shown in Figure 3 .

[0032] 2. Construction of pmRosa26-CAG-hSCARB2 Targeting Vector The intermediate vector pCAG-GFP-N1-Rosa26 LHA was digested with restriction endonuclease Hind3, and the 6 kb fragment was recovered as a ligation vector for later use. The fragment hSCARB2 was amplified using primers hscarb2-GF hscarb2 and hSCARB2-R hSCARB2, the fragment wpre-polyA was amplified using primers wpre-F hSCARB2 and bGHpolyA-R hscarb2, and the fragment Rosa26 RHA was amplified using primers mRosa26-RHA-F hscarb2 and mRosa26-RHA-GR hscarb2. The primer sequences are shown in Table 1. Finally, pCAG-GFP-N1-Rosa26 LHA (Hind3) (6kb), fragment hSCARB2 (1.4kb), fragment wpre-polyA (860bp), and fragment Rosa26 RHA (1.8kb) were connected in one step using the pEASY®-Basic Seamless Cloning and Assembly Kit to successfully construct the pmRosa26-CAG-hSCARB2 Targeting Vector. The vector plasmid map is shown in Figure 4. Targeting Vector pmRosa26-CAG-hSCARB2 Targeting Vector Bacterial liquid identification gel electrophoresis diagram is shown in Figure 5 Three pairs of primers were used for colony identification, namely wpre-F hSCARB2+bGH polyA-R hscarb2, with a product size of 860 bp; H11-hPSGP-jcf2+hSCARB2-R hSCARB2, with a product size of 1512 bp; and H11-RHA-jcR1+mRosa26-RHA-F hscarb2, with a product size of 2449 bp. The primer sequences are shown in Table 1: the sequence of rosa-LHA-spe1F is: SEQ ID NO.1; the sequence of rosa-LHA-spe1R is: SEQ ID NO.2; the sequence of Rosa-LHA-jcF2 is: SEQ ID NO.3; the sequence of rosa-LHA-jcR2 is: SEQ ID NO.4; the sequence of bGHpolyA-R hscarb2 is: SEQ ID NO.5; the sequence of wpre-F hSCARB2 is: SEQ ID NO.6; the sequence of hscarb2-GF hscarb2 is: SEQ ID NO.7; the sequence of hSCARB2-R hSCARB2 is: SEQ ID NO.8; the sequence of mRosa26-RHA-F hscarb2 is: SEQ ID NO.9; the sequence of mRosa26-RHA-GR hscarb2 is: SEQ ID NO.10; the sequence of H11-hPSGP-jcf2 is: SEQ ID NO.11; the sequence of H11-RHA-jcR1 is: SEQ ID NO.12.

[0033] According to the results of colony identification, 12# was sent for sequencing, and the sequence was found to be completely correct after comparison. Figure 6 Therefore, the 12# bacterial solution was extracted using the EndoFree Plasmid Maxi Kit (10) and stored at -80°C for microinjection.

[0034] Table 1 Primer sequences

[0035] Example 2: Construction of KI mice Main reagents: M2 Embryo Media (Cat. No.: MR-015, Sigma, USA); tri-gas incubator (FORMA, USA); dissecting microscope and inverted microscope (NIKON, Japan); micromanipulation system (EPPENDORF, Germany); culture dishes and Pasteur pipettes (FALCON, USA); pipettes (EPPENDORF, Germany); urinary gonadotropin for injection (PMSG) and human chorionic gonadotropin for injection (HCG) were purchased from Ningbo Sansheng Pharmaceutical; mineral oil (Sigma, USA).

[0036] 1. Preparation of recipient mice ICR mice aged 6-8 weeks and weighing 22-28 g were selected as recipient mice. They were mated with vasectomized ICR mice the night before transplantation. The next day, female mice with sperm plugs were selected as recipients for transplantation.

[0037] 2. Ovulation induction in mice Select 4-8 week old C57bl / 6J female mice and inject 5 international units (IU) of pregnant mare serum gonadotropin (PMSG) intraperitoneally. About 48-54 hours later, inject 2.5-5.0 IU of human chorionic gonadotropin (hCG) into the same mice intraperitoneally. Then, the mice are caged together with sexually mature 8-12 week old male C57bl / 6J mice.

[0038] 3. Collection of fertilized eggs The next day after the cage was put together, the mice with sperm plugs were selected to collect fertilized eggs. The cumulus complex was digested with hyaluronidase, and then rinsed with M2 culture medium. The fertilized eggs were placed in M2 culture medium, covered with mineral oil (Sigma, catalog number: M8410-1L), and cultured in an incubator at 37°C and 5% CO2.

[0039] 4. Microinjection During the incubation process of the fertilized eggs, pay attention to the formation of pronuclei. Generally, after the fertilized eggs are cultured for 2-4 hours, obvious pronuclei appear. At this time, Cas9 mRNA (final concentration 25 ng / μl), sgRNA targeting human scavenger receptor class B member 2 (hSCARB2) (final concentration 12.5 ng / μl) and pmRosa26-CAG-hSCARB2 TargetingVector plasmid obtained in Example 1 (10 ng / μl) are co-injected into the pronucleus of the fertilized eggs. After the injection, the fertilized eggs are moved to another fresh M2 culture medium and cultured for 2-3 hours.

[0040] 5. Fallopian tube transplantation 5.1 The transplantation is performed using a glass capillary with a caliber of 150-160μm and a length of about 7-10cm. First, aspirate about 0.05cm of M2 liquid, then aspirate a 0.05cm section of air, then aspirate about 0.05cm of M2 liquid, then aspirate a 0.05cm column of air. After completing two liquid columns and two air columns, aspirate 15 fertilized eggs, then aspirate a 0.05cm column of air, and the last section is a 0.05cm section of M2 liquid. That is, the order of the entire transplantation liquid column is: M2 liquid column-air column-M2 liquid column-air column-embryo liquid column-air column-M2 liquid column; 5.2 The pseudo-pregnant female mice were anesthetized with 1.25% Avertin at a dose of 0.2 mL / 10g intramuscular injection and placed on a 37°C constant temperature pad. The back of the mouse was wiped with iodine cotton and then wiped with 70% alcohol. A small vertical incision (0.3-0.5 cm) was made about 0.5 cm to the left of the midline, between the dorsal hump and the hip joint of the hind leg. The tissue was bluntly separated with ophthalmic forceps, and the fat pad, ovary and fallopian tube were gently removed. The fat pad was fixed with a small spring clip. The constant temperature pad was moved so that the mouse was under the dissecting microscope, and the microscope and mouse position were appropriately adjusted to make the bulge of the fallopian tube clearly visible. Use small ophthalmic scissors to cut a small hole of about 0.02cm in the transparent capsule of the infundibulum, and gently insert the prepared transplant tube from this hole into the fallopian tube to a depth of about 0.1-0.2cm. Gently blow the fertilized egg into the infundibulum, and slowly retract the transplant tube when 3 bubbles are seen in the infundibulum. Then remove the spring clamp and use blunt forceps to restore the fat pad and fallopian tube to their original positions, suture the wound, and apply erythromycin eye ointment on the wound after suturing. Transplant the right fallopian tube of the mouse in the same way.

[0041] After transplantation, the mice were awoke on a constant temperature pad and then transferred to their original cages for maintenance.

[0042] 5.3 Mouse genotype identification Seven days after birth, DNA was extracted from the tail tips of the mice obtained in Example 2, and genotype identification was performed by PCR. The results showed that among the 35 newborn mice, 14 first-generation KI mice (F0) with the hSCARB2 gene were successfully obtained. The male first-generation KI mice No. F0-1 were selected and mated with WT mice to obtain 16 F1 mice. Genotype identification by PCR showed that 7 were positive ( Figure 7 ), and the PCR product was sent to a sequencing company for sequencing. The results showed that the target band sequence was completely consistent with hSCARB2 ( Figure 8 ). The mice were euthanized 10 days after birth, and Western blot analysis showed that hSCARB2 protein was expressed in most tissues of the F1 generation of mice, including the brain, heart, liver, spleen, stomach, and small intestine ( Fig. 9). This indicated that hSCARB2-KI mice were successfully constructed.

[0043] Example 3: Establishment of Coxsackievirus A16 (CVA-16) infection model 1. KI mice and wild-type mice (WT mice) at the age of 10, 21, and 30 days were intracranially injected (ic) with 1*10 7 PFU / ml of CVA-16.

[0044] 2. This application used RT-qPCR to detect the viral load in the heart, liver, lung, brain and hind limbs of mice 5 days (5dpi), 7 days (7dpi) and 10 days (10dpi) after CVA-16 infection. The results showed that viral replication was observed in the brain, heart, liver, lung and hind limb tissues of 10-day-old KI mice 5 days and 7 days after viral infection. As the infection continued, the viral load reached a peak on the 7th day after infection, and high viral replication was observed in the heart, liver and lung tissues ( Fig.10 A), on the 10th day after infection, mice developed hind limb paralysis and paralysis; high viral loads were observed in the heart, liver, lung, brain and hind limb tissues of 21-day-old mice on the 5th and 7th days after virus infection ( Fig.10 B); High levels of viral replication were observed in the brain and hind limb tissues of 30-day-old mice on days 5 and 7 after virus infection ( Fig.10 C). The common feature of KI mice of different ages after virus infection is that on the 7th day, higher viral loads were detected in the brain and hind limbs, which was significantly different from that of WT infected mice (p<0.01). Fig.10 A, B, C), indicating that the distribution of CVA-16 virus in various tissues was consistent with the successful infection and replication in hSCARB-KI mice, and the infection age could be extended to 30 days.

[0045] 3. Through histopathological examination, HE staining results showed: 3.1 KI mouse brain tissue showed moderate edema, sparsely arranged neurons; a large number of neuronal cells were necrotic, nuclear fragmentation, disordered arrangement of neurons, accompanied by a large number of microglial infiltration; a large number of neurons were condensed and darkly stained, with unclear nuclear and cytoplasmic boundaries, and increased basophilia ( Fig.11 , KI); In the brain tissue of WT mice, the neurons in each area have regular morphology, neatly and densely arranged, with large and round nuclei, less chromatin, obvious nucleoli, and no obvious abnormalities ( Fig.11 , WT).

[0046] 3.2 Diffuse myocyte necrosis, nuclear fragmentation, disordered muscle fiber arrangement, and massive lymphocyte infiltration were observed in the hind limb muscle tissue of KI mice ( Fig.11 , KI); Muscle cells can be seen in the cross-section and longitudinal sections of the hind limb muscle tissue of WT mice. The muscle cells in the longitudinal section are slender and cylindrical cells. The sarcoplasm contains many myofibrils arranged parallel to the long axis of the cell. The nucleus is located near the sarcolemma at the periphery of the cell and is elliptical; the cross-section shows several muscle fiber bundles with clear boundaries. The muscle fibers in the muscle bundles are closely arranged, and most of them have an angular appearance. Typical light and dark alternating horizontal stripes can be seen, and no obvious abnormalities are found ( Fig.11 , WT).

[0047] Therefore, the experiment of intracranial injection of CVA-16 virus showed that the gene-edited mice we constructed can be successfully infected with CVA-16. Importantly, 10-day-old mice showed severe symptoms of hind limb paralysis and death after infection, and the virus infection can continue to 30 days.

[0048] The scheme of this application is based on the plasmid pCAG-GFP-N1 as the basic framework. First, the left homology arm LHA of the Rosa26 site is connected to the plasmid pCAG-GFP-N1 to successfully construct the plasmid pCAG-GFP-N1-Rosa26 LHA. Then, the Rosa26-RHA and hSCARB2 fragments and the wpre-polyA fragment are amplified by high-fidelity enzyme PCR and recovered for use; Hind3 is used to cut pCAG-GFP-N1-Rosa26 LHA and the large fragment is recovered as a connection vector for use, and then the three fragments are successfully constructed into the vector using the Gibson Assembly one-step method, that is, the desired plasmid pmRosa26-CAG-hSCARB2 Targeting Vector ( Figure 1 ). To verify the genotype of the mice, the genome extracted from the mouse tail was analyzed by PCR. The results showed that among the 35 F0 mice, 14 KI mice (F0) with the hSCARB2 gene were successfully obtained using CRISPR / Cas9 editing technology. The first male KI mouse No. 1 (F0-1) in the F0 generation was selected to mate with WT mice. After PCR identification, 7 heterozygous positive mice were obtained among the 16 F1 mice. Figure 7 (The mice marked as 1.1, 1.2, 2.1, 3.1, 3.2, 5.1, and 5.2 are heterozygous positive mice). Both sequencing verification and WB identification were positive ( Figure 8 , 9 ), so the mouse was named B6- Gt(Rosa)26Sor em1(CAG -hSCARB2)Knock-in mice (hSCARB2-KI mice for short). And this hSCARB2-KI mouse can be successfully infected with CVA-16 virus. This model showed serious phenomena such as hind limb paralysis and death for the first time. It can be used to study the effects of CVA-16 on the nervous system (CNS); it can also be used to study the pathogenic mechanism of CVA-16 and evaluate CVA-16 vaccines.

[0049] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for constructing a mouse model infected with Coxsackievirus A16, characterized in that: The construction method comprises the following steps: 1) Using the plasmid pCAG-GFP-N1 as the basic skeleton, the left homology arm LHA of the Rosa26 site was connected to pCAG-GFP-N1 to construct the plasmid pCAG-GFP-N1-Rosa26 LHA; 2) Then, the Rosa26-RHA and hSCARB2 fragments and the wpre-polyA fragment were amplified by PCR, and after being digested with Hind3, the large fragments were recovered and used as connection vectors for later use; 3) The large fragment in 2) was successfully constructed into the pCAG-GFP-N1-Rosa26 LHA in 1) using the Gibson Assembly one-step method to obtain the plasmid pmRosa26-CAG-hSCARB2 Targeting Vector; that is, the donor sequence of the target fragment; 4) When the fertilized egg has obvious pronuclei, dilute it with TE buffer, and co-inject Cas9 mRNA, sgRNA targeting hSCARB2, and the donor sequence of the target fragment obtained in 3) into the pronucleus of the fertilized egg; move the injected fertilized egg into another fresh M2 culture medium for culture; 5) After in vitro culture, the cells were transplanted into the oviduct of pseudo-pregnant ICR female mice and raised for delivery; after 20-21 days, F0 generation mice were obtained, and F0 generation positive mice were mated with wild-type mice to obtain FI generation positive mice, that is, hSCARB2-KI mice; 6) CV-A16 was then injected intracranially into the hSCARB2-KI mice, thereby successfully constructing a mouse model infected with Coxsackievirus A16.

2. The construction method according to claim 1, characterized in that: The final concentration of the sgRNA targeting hSCARB2 in 4) is 12.5 ng / μl; the final concentration of the Cas9 mRNA is 25 ng / μl; and the final concentration of the donor sequence of the target fragment is 10 ng / μl.

3. The construction method according to claim 1, characterized in that: The in vitro culture time in 5) is 2-3 hours.

4. The construction method according to claim 1, characterized in that: The construction method also includes: 7) genotyping the mouse model infected with Coxsackievirus A16 constructed in 6) by PCR and WB, detecting the viral load and pathological histological changes by RT-qPCR and HE staining, and the results show that the mouse model infected with Coxsackievirus A16 was successfully constructed.

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