Use of smim19 protein or its encoding gene in resisting porcine epidemic diarrhea virus

Abstract

The present application relates to the technical field of plant breeding, and particularly relates to application of SMIM19 protein or its coding gene in resisting porcine epidemic diarrhea virus. The SMIM19 protein comprises an amino acid sequence as shown in SEQ ID NO. 1, and the coding gene of the SMIM19 protein comprises a nucleotide sequence as shown in SEQ ID NO. 2. The present application finds that the SMIM19 protein has a certain degree of correlation with the porcine epidemic diarrhea virus, and knocking out the coding gene of the SMIM19 protein in cells can effectively improve the resistance of the cells to the porcine epidemic diarrhea virus. The SMIM19 protein and its application provided by the present application can be applied to breeding of animals resisting the porcine epidemic diarrhea virus, or to resisting infection of the porcine epidemic diarrhea virus in the animal breeding industry, which has important significance in the field of animal breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant breeding, and particularly relates to application of SMIM19 protein or its coding gene in resisting porcine epidemic diarrhea virus. BACKGROUND

[0002] In recent years, frequent outbreaks of porcine coronavirus disease have seriously threatened the development process and production efficiency of the pig industry, including various disease types such as porcine infectious gastroenteritis, porcine epidemic diarrhea, porcine delta coronavirus disease, porcine respiratory coronavirus disease, porcine acute diarrhea syndrome coronavirus disease and porcine hemagglutinating encephalomyelitis virus disease.

[0003] Porcine epidemic diarrhea (PED) is a disease caused by porcine epidemic diarrhea virus (PEDV), which is widespread in the world and has brought huge economic losses to the pig industry. Due to the strong variation ability of coronavirus, it is difficult to provide long-term immunity and safe protection for pigs by vaccination. Therefore, from the perspective of the host, using disease-resistant breeding strategies to obtain host susceptible / resistance genes to viruses has become one of the key points to be studied at present. SUMMARY

[0004] In order to solve the problems existing in the prior art, the present application provides application of SMIM19 protein or its coding gene in resisting porcine epidemic diarrhea virus.

[0005] In a first aspect, the present application provides application of SMIM19 protein, or its coding gene, or an inhibitor of its coding gene in resisting porcine epidemic diarrhea virus.

[0006] The present application further provides application of SMIM19 protein, or its coding gene, or an inhibitor of its coding gene in preparing a reagent or kit for resisting porcine epidemic diarrhea virus.

[0007] The present application further provides application of SMIM19 protein, or its coding gene, or an inhibitor of its coding gene in animal breeding for resisting porcine epidemic diarrhea virus.

[0008] Further, by reducing the expression level of SMIM19 protein, an animal species with high resistance to porcine epidemic diarrhea virus is obtained; or animals with inhibited expression level of SMIM19 protein are crossed with other strains to obtain an animal species with high resistance to porcine epidemic diarrhea virus.

[0009] Further, the SMIM19 protein comprises any one of the following amino acid sequences:

[0010] (1) the amino acid sequence shown as SEQ ID NO. 1;

[0011] (2) the amino acid sequence of the protein with the same function obtained by substitution, insertion or deletion of one or more amino acids of the amino acid sequence shown as SEQ ID NO. 1.

[0012] Further, the coding gene of the SMIM19 protein comprises any one of the following nucleotide sequences:

[0013] (1) the nucleotide sequence shown as SEQ ID NO. 2;

[0014] (2) the nucleotide sequence capable of encoding the protein with the same function obtained by substitution, deletion or insertion of one or more nucleotides of the nucleotide sequence shown as SEQ ID NO. 2;

[0015] (3) the nucleotide sequence capable of hybridizing with the nucleotide sequence shown as SEQ ID NO. 2 under stringent conditions.

[0016] Further, the animal is a pig.

[0017] Further, the inhibiting factor comprises one or more of gRNA, siRNA, miRNA, antisense RNA or CRISPRi.

[0018] In the second aspect, the present application provides an anti-porcine epidemic diarrhea virus reagent or kit, comprising one or more of the following gRNAs:

[0019] gRNA-A2: CAACCGATGAGACTGCAATC-TGG;

[0020] gRNA-B1: AAGATAAAATGTGGCTCGGC-AGG.

[0021] The present application has the following beneficial effects:

[0022] The present application researches and finds a SMIM19 protein closely related to porcine epidemic diarrhea virus. By knocking out the coding gene of the SMIM19 protein in the cell, the ability of the cell to resist porcine epidemic diarrhea virus can be effectively improved, and the concentration of virus particles in the cell and the expression level of porcine epidemic diarrhea virus M gene and N gene can be reduced. The SMIM19 protein and its application provided by the present application can be applied to anti-porcine epidemic diarrhea virus animal breeding or anti-porcine epidemic diarrhea virus infection in animal breeding industry, which has important practical value in the field of animal breeding. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings described below are only some embodiments of the present application, and all other drawings obtained by those skilled in the art without creative labor based on these drawings also belong to the protection scope of the present application.

[0024] Figure 1 FIG. 1 is a schematic diagram of gRNA targeting region and PCR and Sanger sequencing provided by the embodiment 1 of the present application; wherein A is the targeting region, B is the Sanger sequencing result, and C is the result of PCR detection of SMIM19 expression level of SMIM19-KO knockout cell line and wild type IPEC-J2.

[0025] Figure 2 FIG. 2 is a detection result chart of PEDV virus M gene expression amount of SMIM19-KO knockout cell line and wild type IPEC-J2 cell line at different time points after inoculation of PEDV virus provided by the embodiment 1 of the present application.

[0026] Figure 3 FIG. 3 is a detection result chart of PEDV virus N protein of three groups of repeated SMIM19-KO knockout cell line and wild type IPEC-J2 cell line at 36h after inoculation of PEDV virus provided by the embodiment 1 of the present application.

[0027] Figure 4 FIG. 4 is an indirect immunofluorescence detection result chart of SMIM19-KO knockout cell line and wild type IPEC-J2 cell line at 24h after inoculation of PEDV virus provided by the embodiment 1 of the present application.

[0028] Figure 5 FIG. 5 is a transmission electron microscope detection result chart of SMIM19-KO knockout cell line and wild type IPEC-J2 cell line at 24h after inoculation of PEDV virus provided by the embodiment 1 of the present application. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present application more clear, the technical solutions of the present application will be described clearly and completely below by combining the drawings in the present application. Obviously, the described embodiments are only some embodiments of the present application, and not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor also belong to the protection scope of the present application.

[0030] The experimental methods involved in the following embodiments are all conventional methods in the art if not specifically mentioned, for example, the conditions suggested by the experimental manual in the art or according to the manufacturer's instructions can be referred to.

[0031] The experimental materials and reagents involved in the following examples can be obtained from commercial channels, for example: Example 1

[0032] The present application verifies the relationship between SMIM19 protein and PEDV, specifically including the following processes:

[0033] I. Experimental materials

[0034] 1. Cells, viruses and vectors

[0035] The cell used in the present application is an immortalized pig small intestinal epithelial cell IPEC-J2.

[0036] The PEDV strain used is PEDV classic strain PEDV CV777, which has been widely circulated in the art and can be purchased commercially.

[0037] The gene knockout vector used is pSpCas9(BB)-2A-GFP, purchased from addgene company, item number 48138.

[0038] 2. Reagents

[0039] First-Strand cDNA Synthesis Super Mix (item number: AE301-02; full gold);

[0040] High Fidelity (HiFi) PCR SuperMix II (-dye) (item number: AS131-21; full gold);

[0041] RNA TransMate (item number: E607402; shengong);

[0042] Alexa Fluor 488 labeled goat anti-mouse IgG (H+L) (item number: A0428; biyun).

[0043] II. Experimental methods

[0044] 1. Gene knockout

[0045] The present application uses CRISPR / Cas9 system to construct SMIM19 gene knockout cell lines. Using the gene sequence on NCBI reference genome, 2 gRNAs are designed for the intron region of the gene on the E-Crispr website for large fragment knockout. The gRNA sequence for the gene is as follows, and the knockout site diagram is as follows Figure 1 .

[0046] gRNA-A2: CAACCGATGAGACTGCAATC-TGG;

[0047] gRNA-B1: AAGATAAAATGTGGCTCGGC-AGG;

[0048] The pSD-gRNA Plasmid kit was used to construct a gRNA expression plasmid. The Cas9 plasmid and the constructed gRNA expression plasmid were co-transfected into IPEC-J2 cells, and G418 drug was used for screening after transfection. After 3 days of screening, 96 single cells were selected for expansion culture. The obtained monoclonal cells were verified by PCR and Sanger sequencing.

[0049] The results are shown in Figure 1 The SMIM19 gene in IPEC-J2 cells was successfully knocked out, named SMIM19-KO knockout cell line, and the expression level of SMIM19 gene was significantly reduced compared with wild type IPEC-J2 cells.

[0050] 2、Cell culture

[0051] The IPEC cells (SMIM19-KO knockout cell line) with successfully knocked out SMIM19 gene were quickly thawed in a 37°C water bath. The cell suspension was transferred to a 15 mL centrifuge tube, and an equal volume of complete culture medium was added and mixed gently. After mixing, centrifuge at 1000 rpm for 5 min, discard the supernatant and add an appropriate amount of complete culture medium, mix gently and then plate the cells. The cells were cultured in a constant temperature incubator at 37°C, 95% humidity and 5% CO2.

[0052] The complete culture medium for IPEC-J2 cell line is DMEM containing 10% fetal bovine serum.

[0053] When the cell density reaches more than 95%, discard the culture medium, wash with PBS for 2 times, add 1 mL of 0.25% trypsin for 2-3 min, and add 1 mL of complete culture medium to terminate digestion. The digested cells were collected in a centrifuge tube and centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed with PBS and centrifuged again. The cells were resuspended with complete culture medium and then plated.

[0054] 3、Establishment of PEDV-infected IPEC-J2 cell model

[0055] The IPEC-J2 cells with SMIM19 gene knocked out and the wild type IPEC-J2 cells obtained in the previous step were uniformly plated in 6-well cell culture plates, and when they grew to about 95%, the IPEC-J2 was infected with PEDV at a multiplicity of infection (MOI) of 0.1. The method is as follows: discard the culture solution, wash with PBS for 3 times, add 50 μL PEDV and 250 μL DMEM, incubate in a 37°C incubator for 1.5 h, shake the cell culture plate every 30 min. After incubation, discard the virus solution, add 1 mL DMEM and continue to culture, collect cells and proteins at different time points (12 h, 24 h, 48 h and 72 h) for the next step detection.

[0056] 4. RNA extraction, reverse transcription, and fluorescent quantitative PCR

[0057] 4.1 Total RNA was extracted by Trizol method, and the specific steps are as follows:

[0058] (1) Prepare a 4°C centrifuge in advance. Wash the cells with PBS gently for 3 times, add 1 mL Trizol to each bottle, shake repeatedly, collect the liquid into a 1.5 mL sterile enzyme-free centrifuge tube, shake well and stand for 3 min;

[0059] (2) Add 200 μL of chloroform, shake well, stand for 3 min after the liquid is layered, 4°C, 12000g, centrifuge for 15 min;

[0060] (3) Take the upper layer of colorless liquid to a new sterile enzyme-free 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well after overturning, and place in -20°C for 30 min;

[0061] (4) Centrifuge at 4°C for 10 min at 12000g, discard the supernatant;

[0062] (5) Add 1 mL of pre-cooled 75% ethanol, invert up and down to make the precipitate float, centrifuge at 4°C for 5 min at 7500g;

[0063] (6) Carefully remove the supernatant and place it at room temperature until the precipitate is dry and transparent;

[0064] (7) Add 30 μL of Nuclear Free water, incubate at room temperature for 10 min,

[0065] (8) Use Agilent Bioanalyzer 2100 to detect the integrity (RIN value) of the sample; and verify the band distribution by 1% agarose gel electrophoresis, and detect the RNA quality and concentration by NanoDrop.

[0066] 4.2 The qualified RNA sample was used for reverse transcription reaction, and the operation was as follows according to the operation instruction of PrimeScript RT reagent Kit with gDNA Eraser: TM The operation was as follows:

[0067] (1) The potential genomic DNA contamination in the sample was removed, and the system was as shown in Table 1, and the reaction condition was 8 min at room temperature, and the next step was performed after the end.

[0068] Table 1 Genomic contamination removal reaction system

[0069] Component Volume gDNA Eraser 1.0 μL 5X gDNA Eraser buffer 2.0 μL RNA 1.0 ng RNase Free dH2O Up to 10.0 μL Total 10.0 μL

[0070] (2) The reverse transcription reaction system was as shown in Table 2, and the reaction condition was 15 min at 37 DEG C and 5 s at 85 DEG C.

[0071] Table 2 Reverse transcription reaction system

[0072] Component Volume Product from previous step 10.0 μL 5X PrimeScript buffer 2 4.0 μL PrimeScript RT Enzyme Mix 1 1.0 μL RT Primer Mix 1.0 μL RNase Free dH2O 4.0 μL Total 20.0 μL

[0073] (3) After the reverse transcription into cDNA, real-time fluorescent quantitative PCR was performed.

[0074] The PCR reaction system 10 ul: SYBP Green Real-time PCR Master Mix 5 uL, upstream and downstream primers (10 uMol / L) 0.5 uL each, cDNA template 1 uL, ddH2O 3 uL.

[0075] The primer list is as shown below. Each sample was repeated for 3 times, GAPDH gene was used as an internal reference, and 2 -ΔΔCt The relative expression amount of PEDV M gene mRNA was analyzed.

[0076] Table 3 Primer information

[0077]

[0078] 5, indirect immunofluorescence

[0079] The IPEC-J2 cells which were passaged for 2 times were inoculated in the 12-well plate with cell climbing sheet at the bottom and placed in a 37℃, 5% CO2 incubator overnight. The IPEC-J2 wild type and SMIM19-KO knockout cell lines were infected with PEDV at MOI = 0.1, and the culture medium was discarded after 12 hours of culture, and PBS was washed for 3 times; 5% BSA was added for room temperature blocking for 30 min; PEDV N protein antibody (1:300 dilution) was incubated, 4℃ overnight. Then PBS was washed for 3 times; the secondary antibody with fluorescent label (1:300 dilution) was added, and incubated at room temperature in the dark, and shaken on the shaking table for 1 hour, and PBS was washed for 3 times; DAPI was added to stain the cell nucleus, and incubated at room temperature in the dark for 15 min, and PBS was washed for 3 times; the anti-fluorescence quencher was used for mounting, and the confocal microscope was observed and photographed.

[0080] 6. Transmission electron microscope observation

[0081] (1) The treated cells were directly centrifuged into a group, and 2.5% glutaraldehyde fixing solution was added along the wall of the centrifugal tube for fixation.

[0082] (2) The sample was rinsed with 0.1M phosphate buffer for 3 times, 15 min each time.

[0083] (3) The sample was fixed with 1% osmium acid for 2.5h.

[0084] (4) The sample was rinsed with 0.1M phosphate buffer for 3 times, 15 min each time.

[0085] (5) The dehydration step was as follows: the sample was treated with 50% ethanol, 70% ethanol, 90% ethanol, 90% ethanol:90% acetone 1:1 mixed solution, 90% acetone in turn, and finally treated with 100% acetone at room temperature, and each process needed to be treated for at least 15 min.

[0086] (6) The sample was embedded: acetone and embedding solution were mixed in a volume ratio of 2:1, treated at room temperature for 3h, then mixed in a volume ratio of 1:2, and embedded overnight; finally, the pure embedding solution was treated at 37℃ for about 3h.

[0087] (7) The solidification process: 37℃ oven overnight, 45℃ oven for 12h, 60℃ oven for 48h.

[0088] (8) The sample was cut into 70nm slices by an ultramicrotome, 3% uranyl acetate-citric acid lead double staining, and observed under a transmission electron microscope.

[0089] III. Results

[0090] The results are shown in the following table: Figure 2 - Figure 5

[0091] Figure 2 ​is the expression amount detection result of the PEDV virus M gene of the SMIM19-KO knockout cell line and the wild type IPEC-J2 cell line of the application at different time points after inoculating the PEDV virus, from which it can be seen that the expression level of the PEDV virus M gene in the SMIM19-KO knockout cell line is significantly lower than that in the wild type IPEC-J2 cell line.

[0092] Figure 3 is the PEDV virus N protein detection result of the three groups of repeated SMIM19-KO knockout cell lines and the wild type IPEC-J2 cell line of the application at 36h after inoculating the PEDV virus, from which it can be seen that the expression level of the PEDV virus N protein in the SMIM19-KO knockout cell line is significantly lower than that in the wild type IPEC-J2 cell line at 36h after inoculating the PEDV virus.

[0093] Figure 4 is the indirect immunofluorescence detection result of the SMIM19-KO knockout cell line and the wild type IPEC-J2 cell line of the application at 24h after inoculating the PEDV virus, from which it can be seen that the expression level of the PEDV virus N protein in the SMIM19-KO knockout cell line is significantly lower than that in the wild type IPEC-J2 cell line at 24h after inoculating the PEDV virus.

[0094] Figure 5 is the transmission electron microscope detection result of the SMIM19-KO knockout cell line and the wild type IPEC-J2 cell line at 24h after inoculating the PEDV virus, from which it can be seen that the wild type IPEC-J2 cell line is slightly edematous, the cell membrane is partially damaged, the intracellular matrix is uniform, the organelle is swollen and presents a vacuole-like shape, and there are a large number of virus particles in the cell. The cell membrane of the SMIM19-KO knockout cell line is complete, the microvilli structure on the membrane surface is slender, the intracellular matrix is uniform, and the number of organelles is rich and most of the structures are acceptable.

[0095] From the above results, it can be concluded that after knocking out the SMIM19 gene in the IPEC-J2 cell line, the resistance of the IPEC-J2 cell to the PEDV is significantly improved.

[0096] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the application, and not to limit them; although the application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the application.

Claims

1. The use of SMIM19 protein, or its encoding gene, or a repressor of its encoding gene, in the preparation of reagents or kits for the treatment of porcine epidemic diarrhea virus; The amino acid sequence of the SMIM19 protein is shown in SEQ ID NO.1; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2; The inhibitory factor includes one or more of the following gRNAs: gRNA-A2: CAACCGATGAGACTGCAATC-TGG; gRNA-B1: AAGATAAAATTGTGCTCGGC-AGG.

2. A reagent or kit for combating porcine epidemic diarrhea virus, characterized in that, Including one or more of the following gRNAs: gRNA-A2: CAACCGATGAGACTGCAATC-TGG; gRNA-B1: AAGATAAAATTGTGCTCGGC-AGG.

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