CN110607280A - Application of EMC3 Gene and Its Targeted Knockout Method - Google Patents

Abstract

The invention belongs to the field of biotechnology, and specifically relates to the application of the EMC3 gene and its fixed-point knockout method. The EMC3 gene is modified by gene editing technology, and the bases in the coding region are changed to cause frameshift mutations, thereby obtaining EMC3 gene knockout cells. strain. Experiments have shown that changing the nucleotide sequence of the EMC3 gene in porcine kidney cells (PK‑15) to cause loss of EMC3 protein expression can significantly interfere with the proliferation of Japanese encephalitis virus (JEV), thereby effectively protecting host cells Protected from JEV-infected invasion-induced death. Multiple sequence alignment analysis found that the EMC3 gene sequence is highly conserved among pigs, humans and mice. Therefore, the EMC3 gene can be used as a gene editing target for anti-JE or for the development of anti-JE virus drugs.

Description

Application of EMC3 Gene and Its Targeted Knockout Method

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of EMC3 gene and its fixed-point knockout method.

Background technique

Various infectious diseases caused by pathogenic microorganisms have always been a major obstacle to the efficient development of the global pig industry. The economic loss of pig farming caused by infectious diseases in China is estimated to be as high as 40 billion yuan every year. Especially since 2018, African swine fever has been introduced into China, which has severely damaged the domestic pig industry. China is the world's largest pig breeding and pork consumer. The epidemic not only affects the pig industry, but also affects related international trade. How to effectively prevent and control the occurrence of diseases has always been the focus of animal husbandry. Although vaccination has played an important role in prevention and control, it has not been able to completely control and eliminate the prevalence of infectious diseases. In the long run, the use of genetic strategies can improve the resistance of pigs to pathogens genetically, so the research on disease resistance breeding has the effect of curing the root cause. With the maturation of molecular biology techniques, a series of progresses have been made in disease-resistant breeding research. For example, candidate genes such as interferon gene, MX1, defensin, NRAMP1 and MHC have been found to be used in disease-resistant breeding. In particular, the rise of CRISPR/Cas9 technology, combined with somatic cell nuclear transfer technology or fertilized egg microinjection technology, has successfully changed the pig CD163 and ANPEP genes, respectively, and prepared a vaccine that can effectively resist porcine blue ear virus (PRRSV) or porcine infectious gastrointestinal The disease-resistant pigs infected and replicated by TGEV provide an important reference for genome editing disease-resistant breeding. Although the disease-resistant genes of some pathogenic microorganisms have been identified, there are many types of bacteria or viruses that can infect pigs, so it is still necessary to strengthen research efforts and carry out continuous disease-resistant breeding research.

Japanese encephalitis (JE) is a zoonotic infectious disease caused by Japanese encephalitis virus (JEV), which is transmitted by insects and mosquitoes. JEV is mainly transmitted in the pig-mosquito-human cycle, in which pigs are an important host for the expansion and storage of JEV, infected mosquitoes transmit JEV to humans through bites, and humans are the final host. Most of the patients with JEV infection are occult infection, and irreversible brain inflammation and severe neurological damage will remain even after the cure. JEV-infected pigs are generally sporadic and recessively infected, which can cause abortion in pregnant sows, acute orchitis or infertility in boars, etc., and bring greater economic losses to the pig industry. The invasion of cells by JEV is a prerequisite for inducing Japanese encephalitis, and the elucidation of the mechanism of invasion will provide more ways for the prevention and treatment of Japanese encephalitis. Therefore, controlling the epidemic of Japanese encephalitis can not only reduce the loss of pig industry, but also has great significance to human health.

In recent years, research on the mechanism of JEV infecting cells has been in-depth. At present, it has been found that JEV can infect various types of cells from pigs, mice and humans, such as PK-15 cells, U251 cells, MDSCs cells, Vero cells, Neuro2a cells and HEK293 cells. There are three steps: body recognition and binding, entry into the host cell through the endocytic pathway, and uncapsid release of nucleic acid. However, the way JEV infects host cells is not yet clear. Therefore, research on the interaction between JEV and the host will help to better understand the infection mechanism of JEV, help to develop new antiviral drugs or carry out gene editing and disease-resistant breeding.

At present, a variety of technologies can be used to study the interaction mechanism between viruses and hosts. The newly developed genome-wide CRISPR/Cas9 library knockout strategy is one of the powerful ways to screen disease-resistant genes. With the advantages of low cost, convenient operation and high efficiency, CRISPR/Cas9 technology has become a powerful helper for gene function research. )” followed the “third-generation genome-directed editing technology”. Compared with the previous two generations of technologies, the biggest breakthrough of CRISPR/Cas9 technology is that it can not only edit a single gene, but more importantly, it can edit multiple genes at the same time, which also provides an effective method for genome-wide screening. The main process of this technology is to design and construct an sgRNA library targeting the entire genome of the species, package the lentivirus and infect the cell line stably expressing Cas9, then use flow cytometry to enrich the positive mutant cell pool, and then use the virus to infect the wild type respectively And the mutant cell library, enrich the surviving cells for high-throughput sequencing to identify the screened sgRNA, and map the corresponding host factors.

The present invention takes the EMC3 gene as the research object, constructs the EMC3 gene editing cell line through the CRISPR/Cas9 technology, and combines a series of molecular and virological experiments to prove that the EMC3 gene is knocked out in pig kidney cells (PK-15), and the deletion EMC3 protein expression can significantly interfere with JEV replication. According to the literature survey, there is no research report on the involvement of EMC3 gene in mediating JEV replication. Therefore, the present invention provides new materials for the research and development of anti-JEV infection drugs and the research on disease-resistant breeding of pigs.

Contents of the invention

The purpose of the present invention is to provide a new application of the EMC3 gene, the deletion of the expression of the EMC3 protein can significantly reduce the replication ability of JEV in the host cell, and the gene can be used as a gene editing target for anti-Japanese encephalitis or for anti-Japanese encephalitis Development of antiviral drugs.

Another object of the present invention is to provide a method for site-specific knockout of the EMC3 gene, using CRISPR/Cas9 lentivirus and flow sorting technology to perform frameshift mutation on the protein coding sequence of the EMC3 gene, so that the expression of the EMC3 protein is lost, thereby preparing EMC3 Gene knockout cell lines.

In order to achieve the above object, the present invention takes the following technical measures:

The application of the EMC3 gene, specifically, the use of genetic engineering techniques (such as ZFN, TALEN and CRISPR/Cas9 technology, etc.) to delete and express the EMC3 protein, thereby inhibiting the replication of JEV in the host cell.

Preferably, the CRISPR/Cas9 system is used to knock out the EMC3 gene, and the sgRNA targeting sequence used is the first exon of the EMC3 gene.

The EMC3 gene is used as a target in the preparation of medicines for preventing and treating animals infected with Japanese encephalitis virus.

Application of the EMC3 gene as a target in the preparation of gene-edited cells or animal models resistant to Japanese encephalitis.

Using CRISPR/Cas9 technology to construct EMC3 gene knockout cell lines, the construction method is as follows:

(1) Use software to design a specific sgRNA targeting the protein coding region of the EMC3 gene, clone the sgRNA fragment into the vector, and construct a target sgRNA expression vector with a GFP screening marker;

(2) Introduce the target sgRNA expression plasmid into cells stably expressing Cas9 protein by lentiviral packaging and infection, sort GFP-positive cells by flow cytometry, and then detect the DNA sequence by TA cloning and Western Blot technology to detect EMC3 protein The expression of EMC3 protein was selected to select the knockout cell line with EMC3 protein deficiency.

Preferably, the nucleotide sequence of the sgRNA is 5'-CGATTGGCAGGACCACCCAGAGG-3', and the PAM sequence contained therein is AGG.

Through the multiple sequence comparison of the nucleotide and amino acid sequences of the EMC3 genes of pigs, humans and mice, it was found that the EMC3 gene sequences from different species are highly conserved, so gene editing of the EMC3 genes from different species can inhibit the replication of JEV .

Compared with the prior art, the present invention has the following advantages:

(1) The present invention comprehensively uses a variety of methods to evaluate the impact of targeted editing of the EMC3 gene on the replication of JEV in host cells. The main technical means used include: using absolute quantification and plaque experiments to detect EMC3 gene knockout cells at different time points The effect of the strain on the copy number and virus titer of JEV-encoded C gene; using immunofluorescence technology to detect the effect of knocking out EMC3 gene on the expression of JEV-encoding NS3 gene; using RTCA real-time label-free cell analysis technology to evaluate the effect of EMC3 gene knockout cells on JEV Induces resistance to cell death. The effect of knocking out the EMC3 gene on the replication of JEV in PK-15 cells was verified by a variety of different experimental techniques from multiple levels, which improved the accuracy of the results.

(2) Japanese encephalitis is a zoonotic infectious disease caused by JEV. After infection, pigs manifested as high fever, abortion, stillbirth and orchitis. Although Japanese encephalitis vaccine is an effective measure to prevent epidemic Japanese encephalitis, because the mechanism of JEV infection is still unclear, there is no effective therapeutic drug, mainly due to the lack of effective molecular targets. Through molecular and virological experimental research, the present invention finds that the EMC3 gene, which is highly conserved in humans, mice and pigs, can be knocked out using gene editing technology, so that the expression of EMC3 protein can be lost, which can significantly inhibit the replication of JEV, and at the same time prove that the EMC3 gene is involved in the replication of JEV key host factors. Therefore, the present invention provides a new target for the development of anti-JEV drugs, the preparation of gene edited cells and animal models.

(3) The present invention also provides a method for efficiently constructing an EMC3 gene knockout cell model, preferably CRISPR/Cas9 technology, providing a specific and efficient sgRNA sequence designed and screened by software, which can be used to efficiently prepare EMC3 protein-deficient cells strain. The method of the invention also provides technical reference and carrier materials for constructing an EMC3 gene knockout animal model.

Description of drawings

Figure 1. Construction of EMC3 knockout cell lines using CRISPR/Cas9 technology

A. A schematic diagram of the sgRNA targeting the first exon of the EMC3 gene designed by software; B. The genotypes of two EMC3 gene-edited cell lines were detected by TA clone sequencing. bp represents base pair, KO represents knockout, - represents the number of bases deleted, + represents the number of bases inserted, (n/m) represents the number of occurrences of the genotype in TA clone sequencing. PAM stands for protospacer adjacent motif. Figure 2. Using Western Blot technology to detect the expression of EMC3 protein in gene knockout cell lines β-Tubulin is tubulin, which is used as a positive internal reference, and WT represents the control group cells that have not been edited by CRISPR/Cas9.

Figure 3. Evaluating the effect of knocking out the EMC3 gene in host cells on JEV replication using plaque assay

WT represents control cells without CRISPR/Cas9 editing, MOCK represents WT cells without JEV treatment, and hpi represents time after virus infection.

Figure 4. Evaluation of the effect of knocking out the EMC3 gene in host cells on the copy number of JEV-encoded C gene by real-time quantitative PCR experiment

WT represents control cells without CRISPR/Cas9 editing.

Figure 5. Immunofluorescence assay was used to detect the effect of knocking out the host cell EMC3 gene on the expression of the JEV-encoded gene NS3

WT represents the control cells without CRISPR/Cas9 editing, DAPI represents the staining of the nucleus, NS3 represents the gene encoded by JEV, and Merge represents the combination of DAPI and NS3.

Figure 6. Comparison of the number of EMC3 knockout cells and wild-type cells after JEV infection for 72 hours using cell growth experiments

WT represents the control cells without CRISPR/Cas9 editing, MOI represents the virus multiplicity of infection, hpi represents the time after virus infection, and EMC3-KO (sorted) represents the GFP-positive cells enriched by flow cytometry after lentivirus infection.

Figure 7. Evaluation of the resistance of EMC3 knockout cell lines to JEV-induced cell death using RTCA real-time label-free cell analysis technology

WT represents the control cells without CRISPR/Cas9 editing, and JEV added represents the addition of JEV-RP9 to the cells at this time point.

Figure 8. Multi-species conservation analysis of porcine EMC3 gene sequence

Detailed ways

The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art, and the reagents or biological materials, unless otherwise specified, have been disclosed.

Example 1: Construction of EMC3 gene knockout cell lines using gene editing technology

First, download the porcine EMC3 gene exon sequence (accession number: ENSSSCG00000011562.3) and the whole pig genome sequence (version number: Sus_scrofa.Sscrofa11.1) from the ensemble database (www.ensembl.org), and then, use sgRNAcas9 The software (www.biootools.com) designed the sgRNA targeting the porcine EMC3 gene (Table 1), and selected the best sgRNA according to the specificity evaluation results. The sgRNA ID was EMC3_A_47, and the target sequence was "CGATTGGCAGGACCACCCAGAGG". There were no off-target sites of 1 and 2 base mismatches (Fig. 1A).

Table 1 Software design and evaluation of sgRNA targeting porcine EMC3 gene

Further, based on the lenti-sgRNA-EGFP lentiviral vector as the backbone, design and synthesize sgRNA primers, as follows EMC3-sgRNA-F: 5′-caccgCGATTGGCAGGACCACCCAG-3′, EMC3-sgRNA-R: 5′-aaacCTGGGTGGTCCTGCCAATCGc-3′, primer After annealing, it was ligated with the linearized lenti-sgRNA-EGFP vector to construct the target plasmid. Concrete reaction system and condition are as follows:

Take 5 μl each of EMC3-sgRNA-F (10 μm) and EMC3-sgRNA-R (10 μm) and vortex mix them, put them on a PCR machine at 95°C for 10 minutes; Cut and purify the recovered linearized lenti-sgRNA-EGFP carrier, mix well, and let stand at 16°C for 30 minutes for ligation.

The ligation product was transformed into competent Escherichia coli DH5a by the heat shock method, and then spread on AMP+ resistant LB solid culture dishes, placed in a 37°C incubator for overnight culture, and selected single clone colonies were expanded and cultured and sent to the company for sequencing. The sequencing primer was U6-F: 5'-ACTATCATATGCTTACCGTAAC-3'. The bacteria liquid successfully identified by sequencing was taken for expansion culture, and the plasmid was extracted using the OMEGA endotoxin-free plasmid extraction kit, which was named lenti-sgRNA-EMC3. Then, package the lenti-sgRNA-EMC3 lentivirus and infect PK-15 cells to construct the EMC3 gene knockout cell line. The specific experimental procedure is as follows:

The day before, inoculate HEK 293T cells in a 10cm ² culture dish, and perform lentivirus packaging when the confluence is 70%-90%. First, take a total of 24 μg of the substance (psPAX2:PMD2.G:lenti-sgRNA-EMC3=1:2:3) and add it to 500 μL of Jetprime Buffer, vortex and mix, then add 40 μL of Jetprime transfectionregent, oscillate to mix, and let Set for 10min. Then, add the above solution into a 10cm ² Petri dish that was replaced with 5ml of 2% FBS medium in advance, and put it back into a 5% CO ₂ incubator at 37°C for cultivation. The medium was changed to 10mL 2% FBS medium at 6h, and 10mL of the same medium was added at 24h, and the supernatant was collected until 60h, centrifuged at 30,000rpm/min at 4°C for 3h, and the supernatant was poured out, and 200μL of pre-cooled Resuspend the lentivirus pellet in PBS, disperse overnight at 4°C and freeze at -80°C.

Take the sgRNA lentivirus targeting the EMC3 gene and infect the PK-15 cell line stably expressing Cas9, culture it for 48 hours, sort the GFP-positive cells by flow cytometry, and sort the single cells to the 96-well culture plate to select the monoclonal cell line . The porcine EMC3 gene sequence was downloaded from the ensemble database, and PCR primers for detecting the sgRNA target region were designed, EMC3-PCR-F: 5′-CCACTTACCCGCCAAGAAGT-3′ and EMC3-PCR-R: 5′-GATGAGAAACCCCGGCAAAC-3′. Use TIANGEN blood/tissue/cell DNA extraction kit to extract monoclonal cell DNA as a template for PCR amplification. The specific PCR reaction system and conditions are as follows:

PCR reaction system:

PCR reaction conditions:

After the PCR amplification is completed, the purified PCR product is connected to the PMD19-T carrier, transformed and plated for culture, and 5 monoclonal colonies are picked each time for expansion and culture, and sent to the company for sequencing to identify the genotype. Comparing the gene sequences of wild-type cell lines, it was found that EMC3-KO#1 and EMC3-KO#16 had a frameshift mutation in the EMC3 gene. As shown in Figure 1, EMC3-KO#1 has 2 forms of base sequence changes, which are deletion of 1 base, deletion of 1 base and insertion of 2 bases; EMC3-KO#16 has only one form of base sequence A base change is a deletion of 1 base.

Further, the expression of EMC3 protein in these two cell lines was detected by Western Blot technology. The specific experimental procedure is as follows:

Inoculate monoclonal cells in a six-well culture plate, and set PK-15 wild-type cells stably expressing Cas9 as the control group. After the confluence reaches about 90%, add 1mM PMSF and 100μl RIPA lysate to each well, and lyse the cells in an ice bath for 30min , centrifuged at 13,000 rpm at 4°C for 20 min, collected the supernatant, and measured the concentration with a BCA protein quantification kit. Take 40 μg of denatured protein samples for polyacrylamide gel electrophoresis, cut the target band according to the molecular weight of EMC3 protein, and cut the β-tubulin protein electrophoresis band as a positive internal reference, and use the wet transfer method to transfer the two groups of proteins at the same time To the PVDF membrane, after blocking with skimmed milk powder, the primary antibody was incubated overnight at 4°C, and developed after incubation with the secondary antibody. As shown in Figure 2, the β-tubulin protein expression of the three cell lines is good, the EMC3 protein is not expressed in the two cell lines EMC3-KO#1 and EMC3-KO#16, but the EMC3 protein expression is good in the wild-type cells, the results show that EMC3 The gene knockout cell line was successfully constructed.

Example 2: Absolute quantification and plaque experiments found that knocking out the EMC3 gene can significantly inhibit the replication ability of JEV in host cells

In order to detect whether knocking out the EMC3 gene can inhibit the replication of JEV in PK-15 cells, absolute quantification and plaque assay were used to detect the effects of two monoclonal cell lines, EMC3-KO#1 and EMC3-KO#16, on the replication of JEV. The specific experimental process is as follows:

First, multiple groups of EMC3-KO#1 and EMC3-KO#16 cells were inoculated at the same time, and PK-15 wild-type cells stably expressing Cas9 were set as the control group. When the confluence reaches about 50%, add the corresponding volume of JEV-RP9 wild-type virus to each well according to MOI=1, shake well and put it back into the cell culture incubator for culture, and change to 2% FBS medium for 2 hours to continue the culture. At different time points such as 12h, 24h, 36h, and 48h, the culture plates were collected and placed at -80°C for two times of repeated freezing and thawing, and the culture medium containing the virus was taken for absolute quantification and plaque experiments.

The day before the plaque experiment, BHK cells were inoculated in a twelve-well culture plate, and inoculation was performed when the confluence reached about 40%. First, the virus culture solution was diluted with DMEM according to the doubling dilution method to obtain virus suspensions of different dilutions; then, 500 μl of each infection BHK cells were taken, and three sets of replicates were set for each dilution. The second hour after inoculation, change the medium to 1ml DMEM (2×) medium containing 2% FBS, 1% double antibody and 50% low-melting point agarose, and let it stand at room temperature. After the medium is completely solidified, put it back into the incubator to continue culturing When obvious plaques are produced, fix with 10% neutral formaldehyde and stain with crystal violet, and calculate the virus titer (PFU/mL) according to the formula of virus titer=plaque number/virus suspension volume/dilution. As shown in Figure 3, at the same dilution, compared with the wild-type control group, the number of plaques produced in each group at 2h was basically similar, indicating that the EMC3 gene had no significant effect on the invasion of JEV; at 12h, 24h and 36h, The plaques produced by EMC3-KO#1 and EMC3-KO#16 cells were significantly reduced, and the virus titer was significantly reduced. Statistical tests found that there were significant differences between the EMC3-KO#1 and EMC3-KO#16 groups and the control group. At 12h The difference is most significant.

200 μl of virus-containing culture solution was taken from each treatment group for absolute quantitative experiments to detect the copy number of JEV. First, use the Takara kit to extract viral RNA, and then reverse transcribe to obtain cDNA. The specific method of reverse transcription is as follows:

1. Removal of Genomic DNA Reaction

The reaction conditions are: 42°C, 2min; 4°C, 2min.

2. Reverse transcription reaction (SYBR Green qPCR method)

The reaction conditions are: 37°C, 15min; 85°C, 5s; 4°C, 2min.

Using the above cDNA as a template, fluorescent quantitative PCR amplification was performed according to the SYRB Green qPCR method. First, specific quantitative PCR primers were designed and synthesized for the C gene encoded by JEV, as follows: JEV-C-F: 5'-GAGCTTGTTGGACGGCAGAG-3' and JEV-C-R: 5'-CACGGCGTCGATGAGTGTTC-3'; The plasmid of the JEV-C gene was used as a template for fluorescent quantitative PCR amplification to obtain the relationship between the Ct value and the copy number, and draw a standard curve; then the experimental samples were amplified, and each group was set up with three replicates. The specific reaction system and conditions of fluorescent quantitative PCR are as follows:

reaction system:

The reaction conditions are:

For the Ct value obtained by fluorescent quantitative PCR, the corresponding virus copy number was calculated according to the standard curve. As shown in Figure 4, at the same dilution, compared with the wild-type control group, the virus copy numbers of each group at 2h were basically similar, indicating that the EMC3 gene had no significant effect on the invasion of JEV; at 12h, 24h, 36h and 48h , the virus copy numbers of EMC3-KO#1 and EMC3-KO#16 were significantly reduced, and statistical tests found that there were significant differences between the EMC3-KO#1 and EMC3-KO#16 groups and the control group, and the difference was the most significant at 12h.

Example 3: Using immunofluorescence experiments to find that knocking out the EMC3 gene can significantly inhibit the expression of JEV-encoded protein in host cells

Furthermore, immunofluorescence assay was used to detect the expression of JEV-encoded gene NS3 at 12 hours after JEV infection of EMC3-KO#1 and EMC3-KO#16 cells. The specific experimental process is as follows:

Inoculate EMC3-KO#1 and EMC3-KO#16 cells, set PK-15 wild-type cells stably expressing Cas9 as a control, and when the confluence reaches about 90%, add a corresponding volume of JEV-RP9 wild-type cells to each well according to MOI=1 Type virus, after shaking well, put it back into the cell incubator for culture, and change to 2% FBS medium in the 2nd hour and continue to cultivate until the 12th hour. The cells at 12h were fixed with paraformaldehyde PFA, treated with 0.3% Tritonx-100, added blocking solution at room temperature for 1h, NS3 (JEV) primary antibody was incubated overnight at 4°C, secondary antibody was incubated at room temperature in the dark for 2h, and then DAPI staining was performed Fluorescence imaging. The results are shown in Figure 5. There was no expression of NS3 protein in wild-type cells not inoculated with JEV, and a large amount of NS3 protein was expressed in wild-type cells inoculated with JEV. NS3 protein in EMC3-KO#1 and EMC3-KO#16 cells inoculated with JEV Not expressive at all. In summary, immunofluorescence experiments demonstrated that knocking out the EMC3 gene significantly affected the expression of the JEV-encoded gene NS3 in PK-15 cells.

Example 4: Observation of the cytopathic effect of JEV infection on EMC3 knockout cell lines

In order to directly observe the lesion effect and cell survival status caused by JEV infection after knocking out the EMC3 gene, RTCA (Real Time Cellular Analysis) and inverted microscope cell imaging technology were used to simultaneously detect.

The effect of JEV inoculated with different MOIs on cell growth was monitored using the inverted microscope cell imaging technique. First, inoculate the GFP-positive cell populations sorted by flow cytometry, that is, EMC3-KO (sorted) cells, and set PK-15 wild-type cells stably expressing Cas9 as a control. When the confluence reaches about 40%, add corresponding volumes of The JEV-RP9 wild-type virus was shaken well and then returned to the cell incubator for culture. The medium was replaced with 2% FBS medium at the 2nd hour and continued to culture until the 72nd hour to observe the cell survival status. The MOI was set to two gradients of 0.03 and 1. As shown in Figure 6, when infected at different MOIs, the survival rate of the cells in the wild-type treatment group was extremely low or almost non-existent, while in the EMC3-KO (sorted) group, a large number of cells survived and almost no death occurred.

RTCA (Roche RTCA DP) was used to monitor the survival status of cells after JEV infection in real time. Inoculate approximately the same number of EMC3-KO#1 and EMC3-KO#16 cells, set PK-15 wild-type cells stably expressing Cas9 as a control, inoculate the corresponding volume of JEV according to MOI=1, and place it on the RTCA instrument every 15 minutes The number of surviving cells was read once, and the cell growth curve was obtained at 60h. WT represents PK-15 wild-type cells stably expressing Cas9, and MOCK represents not inoculated with JEV. The results are shown in Figure 7. The number of cells in the WT-JEV group was extremely reduced at about 20 hours after inoculation with JEV. However, except for the WT-JEV group, the growth of the cells in the other treatment groups was good. The results of two groups of experiments showed that knocking out the EMC3 gene did not affect the normal growth of cells and could resist cell death caused by JEV replication.

Example 5: Analysis of species conservation of EMC3 gene sequence using bioinformatics strategy

From the NCBI Nucleic Acid Database (https://www.ncbi.nlm.nih.gov/), download from pig (accession number: XM_003358517.4), mouse (accession number: NM_175101.3) and human (accession number: NM_018447.3) the mRNA sequence of the EMC3 gene. Enter the mRNA sequence into NCBI's ORF online software (https://www.ncbi.nlm.nih.gov/orffinder/), and its CDS sequence can be extracted, and then use the multiple sequence alignment program CLUSTALW (https://www. genome.jp/tools-bin/clustalw), the results can be seen in Figure 8, the CDS sequence alignment similarity score of pig and human EMC gene is 94.2748, that of pig and mouse is 91.2214, and that of human and mouse for 90.8397. It shows that the EMC gene similarity between pig and human is relatively high. The CDS sequence was translated into a protein-encoded amino acid sequence using ORF software, and multiple sequence alignment analysis was also performed using the CLUSTALW program. It was found that the length of the EMC3 gene protein-encoded amino acid sequence was 261 amino acids, and the similarity between pig and human EMC protein sequences It is 99.6169, that of pig and mouse is 99.2337, and that of mouse and human is 99.6169, indicating that the protein-coding amino acid sequence of EMC3 gene is very conservative, and there is only one amino acid difference. It is speculated that the functions of the EMC3 gene are highly consistent in pigs, humans and mice. Based on this, we predict that the targeted knockout of the EMC3 gene in humans, mice and pigs by gene editing technology can significantly inhibit the replication of JEV in different species of host cells.

Claims (8)

1. The application of the EMC3 gene is characterized in that the protein encoded by the EMC3 gene is deleted, thereby inhibiting the replication of the Japanese encephalitis virus in the host cell. 2. The application according to claim 1, wherein the EMC3 gene is knocked out based on the CRISPR/Cas9 system. 3. The application according to claim 2, wherein the sgRNA targeting sequence adopted by the CRISPR/Cas9 system is the first exon of the EMC3 gene. 4. The application of EMC3 gene as a target in the preparation of medicines for preventing and treating animals infected with Japanese encephalitis virus. 5. The application of the EMC3 gene as a target in the preparation of gene-edited cells or animal models resistant to Japanese encephalitis. 6. the method for site-specific knockout EMC3 gene, is characterized in that, comprises the following steps: (1) Use the software to design a specific sgRNA targeting the coding region of the EMC3 gene, clone the sgRNA fragment into the vector, and construct the target sgRNA expression vector with the GFP screening marker; (2) The target sgRNA expression vector was introduced into cells stably expressing Cas9 protein by lentiviral packaging and infection, and GFP-positive cells were sorted by flow cytometry, and then the DNA sequence was detected by TA cloning and Western Blot technology was used to detect EMC3 According to the expression of the protein, the gene knockout cell line with EMC3 protein deletion was selected. 7. The method for site-directed knockout of EMC3 gene according to claim 6, characterized in that the nucleotide sequence of the sgRNA used is 5'-CGATTGGCAGGACCACCCAGAGG-3'. 8. the EMC3 gene knockout cell line prepared by the method described in claim 6.