Construction method and application of coxsackie virus susceptible animal model

Abstract

The invention provides a construction method and application of a coxsackie virus susceptible animal model, and aims to improve the problem that coxsackie virus is not easy to feel in a conventional animal model, high expression of hKREMEN1 gene on mouse pulmonary epithelium is realized by using a gene editing technology, and the coxsackie virus susceptible animal model is obtained by constructing a mouse stably expressing an hKREMEN1 receptor. The infection ability of the coxsackie virus on mice is improved, a good foundation is laid for research and development of hand-foot-and-mouth disease vaccines, and vaccine preparation and effectiveness evaluation are facilitated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to a method for constructing a coxsackie virus susceptible animal model and application thereof. BACKGROUND

[0002] Hand-foot-mouth disease is a highly infectious disease in infants under the age of 6, with typical symptoms of blisters on the hands, feet, mouth and other parts. In severe cases, it can cause herpangina, myocarditis, encephalitis, and even death. There are many types of enteroviruses that cause hand-foot-mouth disease, among which enterovirus A71 (EV-A71), coxsackie virus A16 (CVA16), coxsackie virus A6 (CVA6), and coxsackie virus A10 (CVA10) are the main pathogenic agents.

[0003] Before the EV-A71 vaccine was launched in 2016 and widely vaccinated, EV-A71 was the core pathogen causing hand-foot-mouth disease, especially severe cases. With the popularization of the EV-A71 vaccine, the epidemiological characteristics of hand-foot-mouth disease have fundamentally changed, and coxsackie viruses such as CVA6 and CVA10 have become the dominant pathogens of current hand-foot-mouth disease, and there is a trend of coexistence of multiple enteroviruses, posing new challenges to the overall prevention and control of hand-foot-mouth disease.

[0004] Currently, the treatment of hand-foot-mouth disease mainly relies on classic antiviral therapies such as interferon and ribavirin, but vaccines are still the most effective means of preventing the disease. The research and development strategies for hand-foot-mouth disease vaccines include inactivated, attenuated, subunit, virus-like particles (VLP), and nucleic acid vaccines, among others. The EV-A71 vaccine has been successfully applied in clinical practice and has played a key role in preventing hand-foot-mouth disease caused by EV-A71. However, the development of coxsackie virus vaccines such as CVA16, CVA6, and CVA10 has long been stuck in a bottleneck: Firstly, the CVA16 inactivated vaccine developed based on the experience of EV-A71 vaccine development is difficult to induce protective antibodies in non-human primates (monkeys); Secondly, the inactivated vaccines for CVA6 and CVA10 and VLP vaccines based on yeast and insect expression systems have only completed preliminary evaluation of effectiveness through a new-born wild mouse model, confirming that maternal antibodies have some protective effect, but there is still no mature non-human primate model evaluation data and clinical trial report; Thirdly, the lack of an appropriate susceptible mouse model for evaluating vaccine effectiveness has become a core technical bottleneck restricting the development of coxsackie virus vaccines such as CVA6 and CVA10.

[0005] Identifying the receptors and functions of viruses invading host cells is crucial for vaccine development. On the one hand, isolating and culturing viruses is the first step in vaccine production. Cell-adapted virus strains are not only the core raw materials for vaccine production, but also the key materials for challenge experiments in the evaluation of vaccine protection. Identifying virus receptors and screening suitable isolation / cultivation cells can greatly improve the efficiency of strain isolation and preparation. On the other hand, the binding ability of homologous receptors in different animals to the same virus varies significantly, resulting in completely different infection characteristics of the virus in different animals. Therefore, identifying virus receptors and functions is also the core basis for selecting appropriate animal models to evaluate the effectiveness of vaccines.

[0006] In the prior art, the receptor research of EV-A71 has been relatively systematic. It has been confirmed that scavenger receptor class B member 2 (SCARB2), P-selectin glycoprotein ligand 1 (PSGL-1), annexin II, and heparan sulfate are functional receptors of EV-A71. Among them, SCARB2 and heparan sulfate are also receptors of CVA16. For CVA6, CVA10 and other coxsackieviruses, recent research has only confirmed that KREMEN1 is a specific receptor, and its role in the CVA10 infection process has been preliminarily elucidated. However, no research has revealed how to use this receptor to break through the technical bottleneck of CVA6 and CVA10 vaccine development.

[0007] With the development of gene editing technology, researchers have constructed a human-derived hSCARB2 transgenic / knock-in mouse model. This model can effectively simulate the clinical symptoms, pathological characteristics and inflammatory response of EV-A71 infection, overcoming the inherent defects of wild newborn mouse models. Compared with non-human primate models, it has the advantages of low cost, simple operation and strong repeatability, providing a key tool for EV-A71 vaccine development and pathogenesis research. However, it needs to be clear that hSCARB2 is only adapted to EV-A71 and CVA16, and cannot solve the problem of poor infectivity of CVA6 and CVA10 in ordinary mice. Moreover, the existing hSCARB2 mice do not have tissue-specific expression, which does not match the infection characteristics of CVA6 and CVA10 (respiratory tract is an important infection pathway), and cannot be directly applied to CVA6 and CVA10 vaccine development.

[0008] In summary, there is no susceptible mouse model for CVA6 and CVA10 in the prior art, nor has the technical idea of applying the KREMEN1 receptor to construct a CVA6 and CVA10 infection model been disclosed. There is also no related research on lung tissue-specific expression of hKREMEN1 to improve the infectivity of CVA6 and CVA10. SUMMARY

[0009] In order to overcome the problem that CVA10 and CVA6 viruses are not ideal in common mouse infection, the purpose of the present application is to provide a method for constructing a coxsackie virus susceptible animal model and application thereof, based on the receptor characteristics and infection characteristics of CVA6 and CVA10, a knock-in mouse model with high expression of human hKREMEN1 in lung tissue is constructed, the infection ability of CVA10 and CVA6 viruses in mice is effectively improved, and the effectiveness evaluation of CVA6 and CVA10 candidate vaccines is laid a foundation.

[0010] The purpose of the present application is realized by the following technical solutions: The present application provides a method for constructing a coxsackie virus susceptible animal model, comprising the following steps: Step 1, vector design and construction, and preparation of gRNA and Cas9 protein: (1) design and construct a targeting vector for inserting a human KREMEN1 (hKREMEN1) gene into a mouse genome safe site, wherein the targeting vector comprises a hKREMEN1 gene expression cassette, and the expression cassette comprises a promoter, a loxP sequence, a selection marker element, a Kozak sequence, a hKREMEN1 coding region sequence and a terminator; (2) design and synthesize a gRNA sequence specifically targeting the mouse genome safe site; (3) prepare Cas9 mRNA or Cas9 protein; Step 2, microinjection and hKREMEN1 transgenic mouse identification: (1) co-microinject the hKREMEN1 targeting vector, gRNA and Cas9 components constructed in step 1 into mouse zygotes; (2) transplant the injected zygotes into pseudopregnant female mice to obtain F0 generation transgenic mice; (3) identify positive F0 generation mice by PCR, and obtain stably inherited transgenic mice through breeding; Step 3, breeding and identification of mice with lung tissue specific expression of hKREMEN1: (1) crossbreed the stably inherited transgenic mice obtained in step 2 with lung tissue specific Cre tool mice, and obtain double positive offspring mice through PCR identification; (2) detect the expression level of the hKREMEN1 gene in the lung tissue of the double positive offspring mice by quantitative PCR; (3) perform coxsackie virus infection experiment on the double positive offspring mice to verify their infection adaptability, i.e. to construct a coxsackie virus susceptible animal model.

[0011] Further, the coxsackievirus includes coxsackievirus A10 (CVA10) and / or coxsackievirus A6 (CVA6).

[0012] Further, the sequence of the hKREMEN1 gene fragment is shown in SEQ ID NO. 1.

[0013] Further, the mouse genome safe site is a ROSA26 site.

[0014] Further, the structure of the expression cassette is "CAG promoter-loxP-PGK-Neo-6 SV40 pA-loxP-Kozak-hKREMEN1 CDS-rBG pA".

[0015] The second aspect of the present application provides a coxsackievirus-susceptible animal model, which is a transgenic mouse constructed by the method of the first aspect, and the mouse specifically highly expresses a human KREMEN1 gene in lung tissue.

[0016] The third aspect of the present application provides an application of the coxsackievirus-susceptible animal model of the second aspect in preparing a vaccine for preventing or treating hand-foot-mouth disease.

[0017] Further, the vaccine includes an inactivated vaccine, an attenuated vaccine, a subunit vaccine, a virus-like particle vaccine or a nucleic acid vaccine.

[0018] Further, the application is to use the coxsackievirus-susceptible animal model to evaluate the effectiveness of a candidate vaccine, and the specific operation includes: The susceptible animal model is infected with coxsackievirus by a nasal drop infection method, and the body weight change and disease score are recorded daily to evaluate the protection effect of the candidate vaccine.

[0019] The present application has the following beneficial effects compared with the prior art: 1. Based on the receptor characteristics and infection characteristics of CVA6 and CVA10, the present application constructs a knock-in mouse model that specifically highly expresses a human hKREMEN1 in lung tissue, significantly improves the infectivity of CVA10 and CVA6 viruses in mice, and solves the problem that ordinary mice are not susceptible to such viruses. 2. The coxsackievirus-susceptible animal model constructed by the present application provides basic support for the development of CVA10 and CVA6 related vaccines, expands the application scenarios of the technology: at the same time, the animal model can provide a more susceptible challenge model for subsequent vaccine protection effect evaluation experiments, provide a key tool for the development of CVA10 and CVA6 related vaccines, and promote the perfection of the hand-foot-mouth disease prevention and control technology system. 3、The coxsackie virus susceptible animal model construction method provided by the application is feasible, stable, low in cost, and high in repeatability, and can effectively help improve the hand-foot-mouth disease prevention and control technical system. BRIEF DESCRIPTION OF DRAWINGS

[0020] The application will be further described below in combination with the drawings and examples: Figure 1 Electrophoretogram for mouse genotype identification; Figure 2 Transcription levels of the hKREMEN1 gene in different tissues of the conditional knock-in mice are shown; Figure 3 Infectivity of CVA10 and CVA6 in transgenic mice and wild mice is shown. DETAILED DESCRIPTION

[0021] The examples are provided to better illustrate the application, but the application is not limited to the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above description, and the improvements and adjustments still belong to the protection scope of the application.

[0022] The endpoints of the ranges and any values in this disclosure are not limited to the precise values stated. The ranges and values should be interpreted as approximately between the stated values. For numerical ranges, the endpoints are included between the stated values, the endpoints and individual points are included between the stated values and individual points, and the individual points are included between the stated values. These numerical ranges are specifically disclosed herein.

[0023] The application will be described in detail below through examples. It should be understood that the following examples are only used to exemplarily further explain and illustrate the content of the application, and are not used to limit the application.

[0024] It should be noted that the experimental methods in the following specific examples are all conventional methods, and the reagents and materials are commercially available unless otherwise specified.

[0025] In the following examples, tracRNA was purchased from Integrated DNA Technologies (IDT); AQP5-Cre mice were purchased from SAIYE Biotechnology Co., Ltd.

[0026] Example 1 The present embodiment provides a construction method of a susceptible animal model of coxsackie virus A10 (CVA10) and coxsackie virus A6 (CVA6), comprising the following steps: Step 1, vector design and construction, and preparation of gRNA and Cas9 protein: (1) Vector construction: The full gene of human KREMEN1 (hKREMEN1, NCBI Reference Sequence: NM_032045.5) was synthesized, and the gene sequence is shown in SEQ ID NO. 1:

[0027] The hKREMEN1 fragment was recovered by enzyme digestion and cloned into the Donor vector. Four single colonies were picked and inoculated in resistant culture solution, and after overnight culture, plasmids were extracted by a small plasmid extraction kit, enzyme-digested, and positive clones were picked for sequencing verification to obtain the hKREMEN1 gene fragment.

[0028] The obtained hKREMEN1 gene was inserted into the vector containing the hKREMEN1 gene expression cassette "CAG promoter-loxP-PGK-Neo-6 SV40 pA-loxP-Kozak-hKREMEN1 CDS-rBG pA" at the mouse ROSA26 site to construct the hKREMEN1 targeting vector.

[0029] In the expression of the above-mentioned recombinant plasmid vector, the symbols such as "-" have no special meaning, and the function is to distinguish the functional structure on the vector. Specifically, each part represents: CAG: a promoter; loxP: a flanking sequence recognized by Cre recombinase; hKREMEN1: the name of the target gene; CMV: a promoter; PGK: a promoter; Neo: neomycin; 6 SV40 pA: a terminator; Kozak: a sequence present in the mRNA of eukaryotes that plays an important role in the initiation of translation; rBG pA: a terminator.

[0030] (2) Design gRNA sequence specifically targeting the mouse ROSA26 site. Specifically, crRNA was synthesized by Kingsway and tracRNA was purchased from IDT.

[0031] The sequence of crRNA is shown in SEQ ID NO. 2: 5'-GAACACUAGUGCACUUAUCCGUUUUAGAGCUAUGCUGUUUUG-3'.

[0032] (3) Mix gRNA with Cas9 protein to complete RNP complex assembly.

[0033] Step 2, microinjection and identification of hKREMEN1 transgenic mice: (1) Select 3-4 week old C57BL / 6J female mice, inject pregnant mare serum (PMSG) and chorionic gonadotropin (hcg) respectively, and the interval between the two is 48 h; after injecting HCG, the female mice are mated with adult fertile male mice to fertilize the female mice; the next day, the female mice are euthanized, and the fertilized eggs are collected from the oviduct and placed in a 37°C constant temperature 5% CO2 incubator for standby; the hKREMEN1 targeting vector constructed in step 1, gRNA and Cas9 components are co-injected into the nucleus of the mouse zygote.

[0034] (2) The injected zygote is transferred to the culture medium and placed in a 37°C constant temperature 5% CO2 incubator for 1 h, and then transplanted; the zygote injected with the exogenous gene is transplanted into the oviduct of the surrogate mother mouse on the day of the plug; after transplantation, the surrogate mother mouse is placed in a clean cage and incubated until it wakes up and is returned to the cage for feeding; after successful oviduct transplantation, the female mouse will generally give birth to a baby about 20 days after the operation, and the F0 generation transgenic mouse is obtained.

[0035] (3) After the F0 generation mouse is born for 1 week, the mouse can be numbered by cutting the paw, and PCR identification is performed; then F1, F2 generation is carried out, and stable transgenic mouse ROSA26_hKREMEN1[CKI / CKI] is obtained by breeding.

[0036] Step 3, breeding and preliminary application of lung-specific hKREMEN1 expressing mice: (1) Use the ROSA26_hKREMEN1[CKI / CKI] mouse obtained in step 2 and AQP5-Cre mouse to carry out multiple rounds of crossbreeding, and identify the double-positive offspring mouse hKREMEN1[CKI / CKI]-AQP5[Cre / +] by PCR.

[0037] (2) Use quantitative PCR to detect the expression level of hKREMEN1 in different tissues. Take the heart, liver, spleen, lung, kidney, brain, intestine, spine, forelimb muscle, hindlimb muscle of hKREMEN1[CKI / CKI] mice and hKREMEN1[CKI / CKI]-AQP5[Cre / +] mice, respectively, extract tissue RNA, reverse transcribe into cDNA, and detect the expression level of hKREMEN1 gene by quantitative PCR.

[0038] (3) Perform CVA10 and CVA6 infection experiments on the mouse to verify its infection adaptability, thereby constructing a coxsackievirus susceptible mouse—hKREMEN1[CKI / CKI]-AQP5[Cre / +] mouse.

[0039] Test example ​​The test example first identifies the genotype of the lung-specific hKREMENl-expressing mouse during the breeding process, and the nucleic acid electrophoresis is as follows Figure 1 .

[0040] Next, the hKREMENl [CKI / CKI] mouse was used as a negative control to detect the gene expression level of the lung-specific hKREMENl-overexpressing mouse hKREMENl [CKI / CKI] - AQP5 [Cre / +] obtained in Example 1. First, the RNA in each tissue was extracted using a nucleic acid extraction kit, and was reverse transcribed into cDNA, which was diluted and subjected to 40 cycles of PCR under the conditions of 95°C for 30 sec, 95°C for 5 s, and 60°C for 5 s. The obtained results were subjected to relative quantitative analysis to determine the expression of the target gene.

[0041] From Figure 2 it can be seen that the expression level of hKREMENl in multiple tissues has been improved, but the most significant overexpression is achieved in lung tissue.

[0042] Further, the test example also evaluates the infection of CVA10 and CVA6 in the lung-specific hKREMENl-expressing mouse described in Example 1. The specific operation is as follows: the lung-specific hKREMENl-expressing mouse is infected with CVA10 and CVA6 by nose drops, the body weight and physical condition of the mouse are recorded daily, and the infection effect of CVA10 and CVA6 is evaluated by drawing the body weight change curve and the morbidity score curve. The results are shown in Figure 3 Compared with the control mouse, CVA10 and CVA6 can cause more obvious weight loss and higher morbidity score in the lung-specific hKREMENl-expressing mouse.

[0043] Finally, it should be noted that the above is only used to illustrate the technical solutions of the present application and is not limiting. Although the present application has been described in detail with reference to the preferred arrangement, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for constructing a coxsackievirus-susceptible animal model, characterized by, The method comprises the following steps: Step 1, vector design and construction, and preparation of gRNA and Cas9 protein: (1) designing and constructing a targeting vector for site-specific insertion of a human KREMEN1 (hKREMEN1) gene into a mouse genome safe site, wherein the targeting vector comprises an hKREMEN1 gene expression cassette, and the expression cassette comprises a promoter, a loxP sequence, a screening marker element, a Kozak sequence, an hKREMEN1 coding region sequence, and a terminator; (2) designing and synthesizing a gRNA sequence specifically targeting the mouse genome safe site; (3) preparing Cas9 mRNA or Cas9 protein; Step 2, microinjection and identification of hKREMEN1 transgenic mice: (1) co-microinjecting the hKREMEN1 targeting vector constructed in step 1, gRNA, and Cas9 components into mouse zygotes; (2) transplanting the injected zygotes into pseudopregnant female mice to obtain F0 generation transgenic mice; (3) identifying positive F0 generation mice by PCR, and obtaining stably inherited transgenic mice through breeding; Step 3, breeding and identification of mice specifically expressing hKREMEN1 in lung: (1) crossing the stably inherited transgenic mice obtained in step 2 with a lung tissue-specific Cre tool mouse, and identifying double-positive offspring mice by PCR; (2) detecting the expression level of the hKREMEN1 gene in the lung tissue of the double-positive offspring mice by quantitative PCR; (3) performing a coxsackievirus infection experiment on the double-positive offspring mice to verify their infection adaptability to the virus, i.e., constructing a coxsackievirus susceptible animal model.

2. The construction method of claim 1, wherein, The coxsackievirus comprises coxsackievirus A10 (CVA10) and / or coxsackievirus A6 (CVA6).

3. The construction method of claim 1, wherein, The sequence of the hKREMEN1 gene fragment is shown in SEQ ID NO.

1.

4. The construction method of claim 1, wherein, The mouse genome safe site is a ROSA26 site.

5. The construction method of claim 1, wherein, The structure of the expression cassette is "CAG promoter-loxP-PGK-Neo-6 SV40 pA-loxP-Kozak-hKREMEN1 CDS-rBG pA".

6. A model for a coxsackie virus susceptible animal, characterized in that, The animal model is a transgenic mouse, which is constructed by the method of any one of claims 1-5, and the mouse specifically highly expresses the human KREMEN1 gene in the lung tissue.

7. The use of the coxsackievirus susceptible animal model of claim 6 in the preparation of a vaccine for preventing or treating hand-foot-mouth disease.

8. Use according to claim 7, characterized in that, The vaccine comprises an inactivated vaccine, an attenuated vaccine, a subunit vaccine, a virus-like particle vaccine, or a nucleic acid vaccine.

9. Use according to claim 7, characterized in that, The use is to use the coxsackievirus susceptible animal model of claim 6 to evaluate the effectiveness of a candidate vaccine, and the specific operation comprises: Infecting the susceptible animal model with coxsackievirus by nasal drop infection, and recording the body weight change and disease score daily to evaluate the protection effect of the candidate vaccine.

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