Construction method and application of enterovirus 71 type susceptible transgenic mouse model

Abstract

The invention discloses a construction method and application of an enterovirus 71 type susceptible transgenic mouse model, and belongs to the technical field of biology. The method comprises the following steps: up-regulating or enhancing or improving the content or / and activity of placenta specific protein 8 in a receptor mouse to obtain a mouse overexpressing the placenta specific protein 8, hybridizing the mouse overexpressing the placenta specific protein 8 with a wild mouse to obtain an F1-generation heterozygote mouse, a homozygote mouse obtained after the F1-generation heterozygote mice are hybridized with each other is an enterovirus 71 type susceptible transgenic mouse model. The application has the advantages that not only is the susceptibility of mice to EV71 increased, but also a proper animal model is provided for exploring the important function of Plac8 in the EV71 infection process.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for constructing a transgenic mouse model susceptible to enterovirus 71 and its application. Background Art

[0002] Enterovirus A71 (EV71), belonging to the genus Enterovirus of the Picornaviridae family, is one of the important pathogens causing hand, foot and mouth disease in infants and young children. According to statistics, the patients infected with EV71 are mainly children under 5 years old, and most of the severe patients are under 3 years old, suggesting that the infection and pathogenesis of EV71 in humans are related to the age of the patients. Importantly, the neurological complications caused by EV71 infection are the main reasons for severe patients and death cases, and it has become one of the main enteric pathogens causing severe neurological infections in infants and young children.

[0003] Placenta-Specific 8 (Plac8) is named because it was initially found to be highly expressed specifically in the placenta of mammals. In recent years, studies have found that PLAC8 plays an important role in the process of virus infection. In the study of the susceptibility of EV71 to infants and young children, it was found that the expression level of PLAC8 was negatively correlated with the age of infants, young children and mice. After 1-day-old wild-type neonatal mice were infected with EV71, the expression of PLAC8 in the brain increased significantly. The above research results suggest that PLAC8 is very likely to play an important role in the severe neurological symptoms of infants and young children caused by EV71 infection. Some studies have shown that mice with transient overexpression of PLAC8 were constructed by injecting Plac8 overexpression plasmid into the superficial temporal vein of neonatal mice, and it was reported that overexpression of PLAC8 in the lungs of mice caused more inflammatory lesions in the lungs and brains (Yang et al., 2021). However, the expression levels of PLAC8 in other tissues have not been reported, nor have the changes in the virus content in the brains of mice with transient overexpression of PLAC8 after infection with EV71 and the effects on the blood-brain barrier function been reported. Therefore, mice with transient overexpression of PLAC8 can only be infected with the virus in a limited time and limited tissues and organs, and there will be no systemic infection or the related pathological manifestations observed in clinical EV71-infected patients. Therefore, it is particularly important to establish a mature and stable Plac8 transgenic mouse model to study the role of PLAC8, which is closely related to the development of the body, in the process of EV71 infecting the body. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Plac8 transgenic mouse model. The technical problems to be solved are not limited to the described technical topics, and those skilled in the art can clearly understand other technical topics not mentioned herein through the following description.

[0005] To solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing a transgenic mouse model susceptible to enterovirus 71, comprising the following steps: upregulating or enhancing or increasing the content and / or activity of placental-specific protein 8 in recipient mice to obtain mice overexpressing placental-specific protein 8, crossing the mice overexpressing placental-specific protein 8 with wild-type mice to obtain F1 hybrid mice, and the homozygous mice obtained by crossing the F1 hybrid mice with each other are the transgenic mouse model susceptible to enterovirus 71.

[0007] In the above method, the wild-type mice and recipient mice are mice in which the content and / or activity of placental-specific protein 8 has not been upregulated or enhanced or increased.

[0008] The wild-type mice and recipient mice may specifically be Balb / C.

[0009] The placental-specific protein 8 is any one of the following:

[0010] A1) A protein with an amino acid sequence of SEQ ID NO:1;

[0011] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in A1), having more than 95% identity with the protein shown therein and having the same function;

[0012] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in either A1) or A2).

[0013] The tag protein includes but is not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0014] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the above protein of the present invention by using known methods, such as directed evolution or point mutation. Those nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of the above protein isolated from the present invention, as long as they encode the above protein and have the function of the above protein, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0015] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gapexistencecost, Perresiduegapcost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively and performing a search, the identity of the amino acid sequence can be calculated, and then the identity value (%) can be obtained.

[0016] In this article, the identity of more than 95% can be 95%, 96%, 97%, 98%, or 99% identity.

[0017] In the above method, the up-regulation or enhancement or increase of the content and / or activity of placental specific protein 8 in the recipient mouse includes the following steps: introducing a substance that up-regulates or enhances or increases the content and / or activity of placental specific protein 8 into the recipient mouse.

[0018] In the above method, the introduction of the substance that up-regulates or enhances or increases the content and / or activity of placental specific protein 8 is achieved by inserting a DNA fragment of nucleotides 10822 to 11991 of SEQ ID NO:3 into the genome of the mouse Chromosome 6 (NCBI Reference Sequence: NC_000072.7) between nucleotides 113050184 and 113050183.

[0019] In the above method, the substance is any one of the following:

[0020] B1), a nucleic acid molecule encoding the aforementioned protein;

[0021] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

[0023] In the above method, the nucleic acid molecule described in B1) is the coding gene of placental specific protein 8 derived from a mouse.

[0024] In the above method, the nucleic acid molecule described in B1) is the coding gene encoding the protein with the amino acid sequence of SEQ ID NO:1.

[0025] In the above method, the nucleotide sequence of the encoding gene is nucleotides 91 to 429 of SEQ ID NO:2.

[0026] In the above method, the expression cassette containing the nucleic acid molecule in B2) refers to DNA that can express the protein described above in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all the regulatory sequences necessary for the nucleic acid molecule encoding any one of the above proteins. The regulatory sequences can direct the coding sequence to express any one of the above proteins in a suitable host cell under its compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences should include a promoter and transcription and translation termination signals. To introduce specific restriction enzyme sites into the vector for ligating the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, regulatory sequences with linkers can be provided. The regulatory sequence can be a suitable promoter sequence, i.e., a nucleic acid sequence recognizable by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences mediating protein expression. The promoter can be any nucleic acid sequence having transcriptional activity in the selected host cell, including mutant, truncated, and chimeric promoters, and can be derived from a gene encoding an extracellular or intracellular protein homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of the protein and can direct the encoded protein into the cell secretion pathway. Any signal peptide coding region that can direct the expressed protein into the secretion pathway of the host cell used can be used in the present invention. It may also be necessary to add regulatory sequences that can regulate protein expression according to the growth of the host cell. Examples of regulatory sequences are those systems that can respond to chemical or physical stimulants (including in the presence of regulatory compounds) to turn on or off gene expression. Other examples of regulatory sequences are those that can amplify genes.

[0027] In certain specific embodiments, the expression cassette in B2) of the above method contains a hepatitis virus post-transcriptional regulatory element and a poly(A) sequence.

[0028] The nucleotide sequence of the hepatitis virus post-transcriptional regulatory element (WPRE) is nucleotides 11173 to 11760 of SEQ ID NO: 3, and the nucleotide sequence of the poly(A) sequence is nucleotides 11765 to 11991 of SEQ ID NO: 3.

[0029] In certain specific embodiments, B3) the nucleotide sequence of the recombinant expression vector is SEQ ID NO: 3.

[0030] In certain specific embodiments, the method comprises the following steps:

[0031] (1) Construction of PLAC8 transgenic mice:

[0032] The CDS sequence of the murine Plac8 201 transcript (nucleotides 91-429 of SEQ ID NO: 2) was inserted into intron 1 of the Rosa 26 gene on the murine genome. In addition to the CDS sequence of the murine Plac8 201 transcript, there are a hepatitis virus post-transcriptional regulatory element (WPRE) and a poly(A) sequence located downstream thereof. The nucleotide sequence of the hepatitis virus post-transcriptional regulatory element (WPRE) is nucleotides 11173 to 11760 of SEQ ID NO: 3, and the nucleotide sequence of the poly(A) sequence is nucleotides 11765 to 11991 of SEQ ID NO: 3.

[0033] Among them, the introduction of the hepatitis virus post-transcriptional regulatory element (WPRE) and the poly(A) sequence can enhance the stability and translation efficiency of mRNA.

[0034] In the specific embodiments of the present invention, it is completed by homologous recombination, and in situ knock-in is completed using the CRISPR-Cas9 technique during homologous recombination.

[0035] In the specific embodiments of the present invention, the method comprises the following steps:

[0036] S1. Prepare Plac8 overexpressing heterozygous mice according to the method of the following steps:

[0037] Inject the sgRNA and Cas9 expression plasmid, and the Donor1 plasmid (SEQ ID NO: 3) into fertilized eggs. The sgRNA targets intron 1 of the Rosa26 gene on the murine genome, and the Donor1 plasmid carries an upstream homologous arm and a downstream homologous arm, and the upstream homologous arm and the downstream homologous arm are respectively located upstream and downstream of intron 1 of the Rosa26 gene on the murine genome.

[0038] Among them, the lengths of the upstream homologous arm and the downstream homologous arm can be 1000 - 3500 bp.

[0039] The fertilized eggs after injection were transplanted into pseudopregnant female mice, and the obtained F0 generation chimeric mice were designated as Plac8 / F0 generation chimeric mice.

[0040] The Plac8 / F0 generation chimeric mice were crossed with wild-type mice, and the obtained heterozygous mice were Plac8 heterozygous mice. Continuous intercrossing was carried out, and the obtained homozygous mice were Plac8 homozygous mice.

[0041] (2) Establishment of EV71 infection model:

[0042] The EV71 clinical strain used in this study was amplified in RD cells. 1-day-old Balb / c wild-type and Plac8 transgenic mice were infected with EV71 virus solution through two routes: intraperitoneal injection and intracranial injection. Control mice were injected with an equal volume of RD cell culture supernatant. After intraperitoneal infection, the number of surviving mice in each group was recorded daily for 2 weeks. On the 5th day after intraperitoneal infection and the 3rd day after intracranial infection, mouse brain tissues were taken for detection of viral RNA, expression detection of receptors SCARB2 and PSGL-1, tight junction protein ZO-1, and transcytosis molecule MFSD2A.

[0043] In the above method, the susceptibility of the enterovirus 71-susceptible transgenic mouse model to enterovirus 71 is higher than that of the receptor mice or wild-type mice.

[0044] The present invention also provides the use of the foregoing substance in the preparation of an enterovirus 71-susceptible transgenic mouse model.

[0045] The present invention also provides the use of the foregoing enterovirus 71-susceptible transgenic mouse model in the development or screening of drugs for preventing and / or treating hand, foot, and mouth disease or in the research of hand, foot, and mouth disease.

[0046] The advantages of the present invention are that it not only increases the susceptibility of mice to EV71, but also provides a suitable animal model for exploring the important functions of PLAC8 during EV71 infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a schematic diagram of the construction strategy for transgenic mice.

[0048] Figure 2 It is a schematic diagram of the homologous recombination vector for transgenic mouse construction.

[0049] Figure 3 It is the PCR identification result of the genotype of transgenic mice.

[0050] Figure 4RT-qPCR results of Plac8 mRNA expression.

[0051] Figure 5 Survival rate of Plac8 transgenic mice after EV71 infection.

[0052] Figure 6 Changes in viral RNA in the brain after EV71 infection.

[0053] Figure 7 Changes in the expression of SCARB2 and PSGL-1 in the brain after EV71 infection.

[0054] Figure 8 Changes in the expression of ZO-1 and MFSD2A in the brain after EV71 infection. Detailed implementation mode

[0055] The present invention will be further described in detail below in conjunction with the specific implementation modes. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0056] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0057] In the following embodiments, all quantitative tests are set with three repeated experiments, and the results are averaged.

[0058] The following embodiments use GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and are tested by One-way ANOVA. P < 0.05 (*) indicates significant differences.

[0059] Example 1. Construction of Plac8 transgenic mouse model

[0060] Balb / c was purchased from Beijing SPF Biotechnology Co., Ltd., and the product number is B201-02.

[0061] The construction of transgenic mice is based on the Balb / c research background and is achieved by homologous recombination supplemented by CRISPR-Cas9 site-directed knock-in technology.

[0062] Murine Plac8 is located on chromosome 5 of the murine genome, has 4 transcripts, and 3 protein products. The amino acid sequence of the protein transcribed by the transcript used in the subsequent examples is SEQ ID NO:1, and the nucleotide sequence of the coding gene of this protein is the nucleotide positions 91 - 429 in SEQ ID NO:2.

[0063] In this invention, during homologous recombination, the CRISPR - Cas9 site - directed knock - in technology is used to break double - stranded DNA, thereby improving the homologous recombination efficiency. Finally, the homologous recombination vector containing the Plac8 gene is site - specifically inserted into the murine Rosa26 gene (Chromosome6:113,076,031). Under the action of the CAG promoter, the Plac8 gene is overexpressed ( Figure 1 ).

[0064] 1. Construction of the homologous recombination vector

[0065] The full sequence of the R26 - e(CAG - Plac8 - WPRE - polyA)1 recombination vector is SEQ ID NO:3. The DNA fragment from position 9123 to 11991 in SEQ ID NO:3 is the DNA fragment to be subsequently inserted into the murine Rosa26 gene. The coding sequence of the Plac8 gene 201 transcript (encoding a protein with an amino acid sequence of SEQ ID NO:1, a total of 112 amino acids) is from position 10822 to 11160. The hepatitis virus post - transcriptional regulatory element (WPRE) is from position 11173 to 11760, and poly(A) is from position 11765 to 11991. The rest is the backbone sequence of R26 - e(CAG - WPRE - polyA)1.

[0066] Plac8 is located on chromosome 5, has 4 transcripts and 3 protein products. It is intended to insert the CDS corresponding to Plac8 - 201 into the position of ggacacactaaggga---insert---gcttgggtgatag in the murine Rosa 26 gene (that is, insert the nucleotide positions 9123 to 11991 of the nucleotide sequence SEQ ID NO:3 between the nucleotide positions 113050184 and 113050183 of murine genome Chromosome 6 (NCBI Reference Sequence: NC_000072.7)).

[0067] 2. Design of sgRNA for CRISPR - Cas9 technology and in vitro transcription of sgRNA and Cas9 mRNA

[0068] (1) Design an sgRNA targeting intron 1 of the murine genome Rosa26, and its targeting sequence is as follows:

[0069] 5'-GGGGACACACTAAGGGAGCTTGG-3'.

[0070] Transcribe the target sequence and Cas9 mRNA by in vitro transcription, purify the products after transcription to obtain an sgRNA solution (sgRNA content is 500 ng / μl, the balance is; 96 μl) and a Cas9 mRNA solution (Cas9 mRNA content is 1000 ng / μl, the balance is 500 μl).

[0071] 3. Construct Plac8 transgenic mice

[0072] The specific operations are as follows:

[0073] (1) Prepare F0 generation mice

[0074] Mix the sgRNA solution obtained in step 2, the Cas9 mRNA solution, and the Donor1 plasmid (R26-e(CAG-Plac8-WPRE-polyA)1 solution) obtained in step 1 according to the injection system: 100 μl. 500 ng of Cas9 mRNA, 200 ng of sgRNA, and 1200 ng of Donor1, and then inject the mixture into 200 fertilized eggs of Balb / C mice. Transplant the injected fertilized eggs into 8 pseudopregnant female mice, with each pseudopregnant female mouse injected with 25 fertilized eggs. The mice are born about 20 days later, a total of 9 mice, which are labeled as F0 generation mice. About 7 days after the mice are born, cut their tails, lyse the mouse tails, and use the lysate as a template. Use primer pairs M1 / M2 (sequences are shown in Table 1) and M3 / M4 (sequences are shown in Table 1) to perform PCR detection on the aforementioned 5' homologous arm and 3' homologous arm in the F0 generation mice respectively. The 5' arm homologous recombination positive genome should amplify a 3.4 kb fragment, and the negative genome should amplify a 5.1 kb fragment; the 3' arm homologous recombination positive genome should amplify a 3.6 kb fragment, and the negative genome should amplify a 6.5 kb fragment. Mice with a 5' homologous arm band of 3.4 kb fragment and a 3' homologous arm band of 3.6 kb fragment are positive mice, denoted as F0 generation chimeric mice. The PCR amplification results of M1 / M2 and M3 / M4 show that a total of 3 F0 generation chimeric mice are obtained.

[0075] (2) Prepare F1 generation mice

[0076] Hybridize the F0 generation chimeric mice (3, 1 male and 2 females) with Balb / C wild-type mice (5, 4 females and 1 male) to obtain a total of 23 F1 generation mice. When the F1 generation mice are about 7 days old, cut their tails, lyse the mouse tails to obtain lysates. Using the above lysates as templates, use primer pairs M1 / M2 (sequences are shown in Table 1) and M3 / M4 (sequences are shown in Table 1) to perform PCR detection on the aforementioned 5' homologous arm and 3' homologous arm in the F1 generation mice respectively. The 5' arm homologous recombination positive genome should amplify a 3.4 kb fragment, and the negative genome should amplify a 5.1 kb fragment; the 3' arm homologous recombination positive genome should amplify a 3.6 kb fragment, and the negative genome should amplify a 6.5 kb fragment. Mice with a 5' homologous arm band of 3.4 kb fragment and a 3' homologous arm band of 3.6 kb fragment are positive mice, denoted as F1 generation heterozygous mice.

[0077] (3) Obtain homozygous mice

[0078] Further cross the F1 generation heterozygous mice in (2) (6 female mice and 2 male mice). When the mice are about 7 days old, cut their tails. After lysing the mouse tails, obtain mouse tail lysates. Take the mouse tail lysates as templates and perform PCR detection with primer pairs P1 / P2 (sequences are shown in Table 1) and P3 / P4 (sequences are shown in Table 1) respectively. Primer pair P1 / P2 amplifies a 994 bp band, and no amplification band for P3 / P4 indicates a wild-type mouse. Primer pair P1 / P2 amplifies a 994 bp band, and P3 / P4 amplifies a 602 bp band indicates a heterozygous mouse. Primer pair P1 / P2 has no amplification band, and P3 / P4 amplifies a 602 bp band indicates a homozygous mouse.

[0079] The results are as Figure 3 shown. Figure 3 In the lanes in

[0080] From left to right are M, B, B, 1, 2, 3, 4, 5, 6, 7, 8, 9, M, B, B, 1, 2, 3, 4, 5, 6, 7, 8, 9, M. M is DNAmaker DL2000, and the sizes of the bands from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp in sequence; B is a blank control, representing the PCR experimental results with water as the template. Numbers 1, 2, 3, 4, 5, 6, 7, 8, and 9 represent the results of amplification with primer pair P1 / P2 or PCR amplification with primer pair P3 / P4 using the mouse tail lysates of mice numbered 1, 2, 3, 4, 5, 6, 7, 8, and 9 as amplification templates.

[0080] As can be Figure 3 seen, mice numbered 1 - 3 are wild-type mice, mice numbered 4 - 6 are heterozygous mice, and mice numbered 7 - 9 are homozygous mice ( Figure 3 ).

[0081] Table 1. Detection of Plac8 Gene

[0082] Name Sequence (5’-3’) M1 GCCGGGCCTCGTCGTCTG M2 TGAGGGCAATCTGGGAAGGTT M3 GGGGGAGGGGAGTGTTGC M4 TTCTTCCTGCCTGCCTTCTGTGAC P1 TCAGATTCTTTTATAGGGGACACA P2 TAAAGGCCACTCAATGCTCACTAA P3 AAAGTCCCGGAAAGGAGCTG P4 AACCTGGATTCGTTCGTGCT

[0083] Compared with Balb / C wild-type mice, the following changes occurred in both homologous chromosomes of the aforementioned homozygous mice: The Plac8 transgenic mice were obtained by inserting a DNA fragment of nucleotides 10822 to 11991 of SEQ ID NO:3 into intron 1 of the Rosa 26 gene of wild-type mice (i.e., inserting a DNA fragment of nucleotides 10822 to 11991 of SEQ ID NO:3 between nucleotides 113050184 and 113050183 of Chromosome 6 of the mouse genome), and the obtained transgenic mice were named Plac8 transgenic mice (i.e., mice overexpressing placental-specific protein 8).

[0084] To further verify the expression of the Plac8 gene in the aforementioned homozygous mice, specific primers for the Plac8 gene were designed, and the specificity and sensitivity of the primers were screened by synthesizing plasmids. Subsequently, the expression of Plac8 mRNA was detected by RT-qPCR, and the primer sequences are shown in Table 2. The reaction system of RT-qPCR is as follows: 2×SuperReal PreMixPlus: 12.5 μl, forward primer (10 μM): 0.75 μl, reverse primer (10 μM): 0.75 μl, cDNA template: 1 μl, water: 11 μl, a total of 25 μl. The reaction program of RT-qPCR is as follows: Pre-denaturation: 95°C, 15 min; PCR reaction: 95°C, 10 s, 60°C, 32 s, 40 cycles. Using the mouse-derived GAPDH gene as an internal reference gene, the 2 -ΔΔCt method was used to analyze the RT-PCR results and calculate the relative gene expression level.

[0085] The experimental data were processed using GraphPad Prism 8, and significant analysis was performed using IBM SPSS Statistics 22 statistical software. The comparison between two groups of data was performed using Student's t-test. p < 0.05 indicates significant difference, p < 0.01 indicates very significant difference, and p < 0.001 indicates extremely significant difference.

[0086] The results showed that Plac8 mRNA was expressed in the brain, muscle, intestine, and lung tissues of Plac8 transgenic mice; the expression level of Plac8 was the highest in the brain and relatively high in the muscle and lung ( Figure 4 ).

[0087] Table 2. Primer Sequences

[0088] Name Sequence (5’-3’) Plac8-F ACCTGGATTCGTTCGTGCTC Plac8-F CAGACTCCGCAGTCACTGAA GAPDH-F ATGTTTGTGATGGGTGTGAA GAPDH-R ATGCCAAAGTTGTCATGGAT

[0089] Example 2. Analysis of Susceptibility and Pathogenicity of EV71-Infected Plac8 Transgenic Mouse Model

[0090] 1. Susceptibility of Plac8 Transgenic Mice to EV71

[0091] The EV virus solution (EV-A71) is described in the following non-patent literature: "Jing Xie b,1 , Xinyan Hu a,b,1 , HuanLi b,1 , Hongwei Zhu d , Weishi Lin b , Lizhong Li b , Ji Wang c,*** , Hongbin Song b,** , Leili Jia a,b,* . Murine models of neonatal susceptibility to a clinical strain of enterovirus A71. Virus Res. 2023 Jan 15:324:199038. doi: 10.1016 / j.virusres.2022.199038. Epub 2023 Jan 1.", which is publicly available from the Center for Disease Control and Prevention of the Chinese People's Liberation Army. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0092] Preparation method of EV71 virus solution: Inoculate 100 μl of EV71 virus seed (Genbank ID: KJ004559.1) into a T25 culture flask paved with RD cells, culture at 37°C and 5% CO2, observe the cytopathic effect every day until 70% - 80% of RD cells show agglutination and shrinkage. Freeze-thaw the whole bottle of cells 3 times repeatedly, take the supernatant after centrifugation, and filter it through a 0.22 μm filter to obtain the EV71 virus solution.

[0093] The detection results show that the titer of the virus in the aforementioned EV71 is 5.5×10 6 pfu / mL.

[0094] Evaluate the susceptibility of Plac8 transgenic mice to EV71 according to the following operations. The experiment is repeated 3 times, and each repetition is as follows:

[0095] Plac8 transgenic mouse group: The EV71 virus solution was used to infect Plac8 transgenic mice by intraperitoneal injection at a dose of 80 μl per mouse. There were 10 mice in this group. The survival number of Plac8 transgenic mice was monitored continuously for 14 days. Survival rate = (number of surviving mice / total number of mice) * 100%.

[0096] Wild-type mouse group (control group): Wild-type mice BALB / C were used to replace Plac8 transgenic mice in the Plac8 transgenic mouse group, and the remaining operations were the same as those in the Plac8 transgenic mouse group.

[0097] Blank group: An equal volume of DMEM cell culture medium was used to replace the virus solution in the Plac8 transgenic mouse group, and the remaining operations were the same as those in the Plac8 transgenic mouse group.

[0098] The results are as Figure 5 shown. On the 4th day after infection, death began to occur in Plac8 transgenic mice; on the 9th day after infection, death occurred in wild-type mice, and further death occurred in Plac8 transgenic mice; on the 14th day after infection, the survival rate of wild-type mice was 80%, while the survival rate of Plac8 transgenic mice was only 60%, indicating that Plac8 transgenic mice are more susceptible to EV71 compared with wild-type mice.

[0099] 2. Replication ability of EV71 in the brains of Plac8 transgenic mice

[0100] The replication ability of EV71 in the brains of Plac8 transgenic mice was evaluated according to the following operations. The experiment was repeated 3 times, and each repetition was as follows:

[0101] Plac8 transgenic mouse group: 3 mice were intracranially injected with 30 μl of the EV71 virus solution in "1. Susceptibility of Plac8 transgenic mice to EV71", and 3 mice were intraperitoneally injected with 80 μl of the EV71 virus solution in "1. Susceptibility of Plac8 transgenic mice to EV71". Euthanasia was performed on the 3rd day after intracranial infection and the brain tissues were collected, and euthanasia was performed on the 5th day after intraperitoneal infection and the brain tissues were collected. The virus content in the brain was detected by qPCR, and GAPDH was used as an internal reference (the primer sequences are shown in Table 2). qPCR reaction system: qPCR reaction program:

[0102] EV71-VP1.-F: 5'-CCAATCTCAGCGGCTTGGAG-3'.

[0103] EV71-VP1-R: 5'-CACTCAAGCTCTACCGGCAC-3'.

[0104] SCARB2-F: 5'-AGAAGGCGGTAGACCAGAC-3'.

[0105] SCARB2-R: 5'-CTGTAGGTGTATGGCCCCAC-3'.

[0106] PSGL-1-F: 5'-TTGTGCTGCTGACCATCT-3'.

[0107] PSGL-1-R: 5'-TCCTCAAAATCGTCATCC-3'.

[0108] ZO-1-F: 5'-GATGAGCGGGCTACCTTA-3'.

[0109] ZO-1-R: 5'-TGGAGACTGCGTGGAATG-3'.

[0110] MFSD2A-F: 5'-CTCCTGGCCATCATGCTCTC-3'.

[0111] MFSD2A-R: 5'-GGCCACCAAGATGAGAAA-3'.

[0112] Wild-type mouse group (control group): Replace the Plac8 transgenic mice in the Plac8 transgenic mouse group with wild-type mice BALB / C, and the remaining operations are the same as those in the Plac8 transgenic mouse group.

[0113] Blank group: Replace the virus solution in the Plac8 transgenic mouse group with an equal volume of DMEM cell culture medium, and the remaining operations are the same as those in the Plac8 transgenic mouse group.

[0114] The results are as Figure 6 shown. On the 3rd day after intracranial infection with EV71, the expression level of EV71 in the brains of Plac8 transgenic mice was 4.3 times that of wild-type mice ( Figure 6 ). On the 5th day after intraperitoneal infection, the expression level of EV71 in the brains of Plac8 transgenic mice was 18.9 times that of wild-type mice. The results indicate that regardless of the EV71 infection route, the virus content in the brains of Plac8 transgenic mice is significantly higher than that of wild-type mice, indicating that EV71 can replicate efficiently in the brains of Plac8 transgenic mice.

[0115] Whether it is the intracranial infection or intraperitoneal infection route, compared with wild-type mice, the expression levels of SCARB2 and PSGL-1 receptors in the brains of Plac8 transgenic mice infected with EV71 are increased ( Figure 7) In intracranial infection, the expression level of SCARB2 in the brains of Plac8 transgenic mice was 1.3 times that of wild-type mice, and the expression level of PSGL-1 in the brains of Plac8 transgenic mice was 3.1 times that of wild-type mice, showing significant differences. In intraperitoneal infection, the expression level of SCARB2 in the brains of Plac8 transgenic mice was 1.4 times that of wild-type mice, showing a significant difference; the expression level of PSGL-1 in the brains of Plac8 transgenic mice was 2.4 times that of wild-type mice, showing a significant difference. The results indicate that the overexpression of PLAC8 may enhance the infectivity of EV71 by promoting the expression of SCARB2 and PSGL-1 receptors in the brain.

[0116] Regardless of the intracranial infection or intraperitoneal infection route, compared with wild-type mice, the expression levels of ZO-1 and MFSD2A in the brains of Plac8 transgenic mice infected with EV71 decreased ( Figure 8 ). Thus, it can be seen that the overexpression of PLAC8 may disrupt the transcytosis function of the integrity of the blood-brain barrier and play an important role in the infection of the central nervous system by EV71.

[0117] SEQ ID NO:1

[0118] MAQAPTVIVTQPGFVRAPQNSNWQTSLCDCFSDCGVCLCGTFCFTCLGCQVAADMNECCLCGTTVAMRTLYRTRYG IPGSICDDYMVTLFCPVCSVCQLKRDINRRRAMNAF.

[0119] SEQ ID NO:2

[0120] 5'-ctatttgtagtaagactcaaccccagaccacaggaccggttctgcccaacccttttgaactacttggtctttt gagacctcgcatcgaagATGGCTCAGGCACCAACAGTTATCGTGACTCAACCTGGATTCGTTCGTGCTCCCCAAAATTCCAACTGGCAGACCAGCCTGTGTGATTGCTTCAGTGACTGCGGAGTCTGCCTCTGTGGGACCTTTTGTTTCACTTGTCTTGGATGTCAAGTGGCAGCTGACATGAATGAGTGTTGTCTGTGTGGAACAACGGTGGCCATGAGGACTCTCTACCGAACCCGATACGGCATTCCTGGATCTATTTGTGATGACTACATGGTCACACTCTTCTGTCCTGTTTGCTCTGTGTGCCAACTCAAGAGAGACATTAACAGGAGGAGAGCCATGAACGCTTTCTAAggagctggatggcaagagctctggctgaagaagctcaactcagcacacactccttcagcctgagatttttcaaatctttggcaactgagatgggatggatccatttaattagagaacggtgaaatctttctagttgggctttttgatttattttaaatggatattgctctttgacttggtttcttcttgctcccatatcatcaaatattggagcctataatttttttaccttacattttaggtagaaaccaaataaaagattttgctaagaaga-3'.

[0121] SEQ ID NO:3

[0122]

[0123] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by using conventional techniques known in the art.

Claims

1. A method for constructing a transgenic mouse model susceptible to enterovirus 71, comprising the following steps: upregulating or enhancing or increasing the content and / or activity of placental specific protein 8 in recipient mice to obtain mice overexpressing placental specific protein 8, crossing the mice overexpressing placental specific protein 8 with wild-type mice to obtain F1 hybrid mice, and the homozygous mice obtained by crossing the F1 hybrid mice with each other are the transgenic mouse model susceptible to enterovirus 71.

2. The method according to claim 1, wherein The step of upregulating or enhancing or increasing the content and / or activity of placental specific protein 8 in recipient mice comprises the following steps: introducing a substance that upregulates or enhances or increases the content and / or activity of placental specific protein 8 into recipient mice.

3. The method according to claim 2, wherein The placental specific protein 8 is any one of the following: A1) A protein with the amino acid sequence of SEQ ID NO:1; A2) A protein obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in A1), having more than 95% identity with the protein shown therein and having the same function; A3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in either A1) or A2).

4. The method according to claim 3, wherein The substance is any one of the following: B1) A nucleic acid molecule encoding the protein described in claim 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

5. The method according to claim 4, characterized in that, The nucleic acid molecule described in B1) is the coding gene of placental specific protein 8 derived from mice.

6. The method according to claim 5, characterized in that, The nucleotide sequence of the coding gene described in B1) is the nucleotide positions 91 - 429 in SEQ ID NO:

2.

7. The method according to any one of claims 4 to 6, characterized in that, The expression cassette described in B2) contains a hepatitis virus post-transcriptional regulatory element and a poly(A) sequence.

8. The method according to any one of claims 1-7, characterized in that, The transgenic mouse model susceptible to enterovirus 71 has a higher susceptibility to enterovirus 71 than the recipient mice.

9. Use of the substance described in any one of claims 4 - 7 in the preparation of a transgenic mouse model susceptible to enterovirus 71.

10. Use of the transgenic mouse model susceptible to enterovirus 71 described in any one of claims 1 - 8 in the development or screening of drugs for preventing and / or treating hand, foot and mouth disease or in the research of hand, foot and mouth disease.

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