Application of preparation for knocking out or inhibiting porcine Jagged1 gene expression in resisting porcine epidemic diarrhea virus infection

Abstract

The invention belongs to the field of gene engineering and biological medicine, and discloses application of a preparation for knocking out or inhibiting pig Jagged1 gene expression in resisting porcine epidemic diarrhea virus, and the NCBI login number of mRNA of the Jagged1 gene in a pig is XM021077352.1, or the Jagged1 gene homologous with the gene in the pig. The gene is found to be knocked out in porcine cells LLC-PK1 and ST and can effectively inhibit PEDV proliferation, so that the gene can be used as a novel target for development of drugs for inhibiting PEDV replication and research on disease resistance breeding.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biomedicine, and specifically relates to the application of a preparation for knocking out or inhibiting the expression of pig Jagged1 gene in resisting porcine epidemic diarrhea virus infection. Background Art

[0002] Porcine Epidemic Diarrhea (PED) is a contact enteric infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV). The pigs have symptoms of watery diarrhea, vomiting and dehydration, and the mortality rate of newborn piglets is high. Since it was first discovered in breeding pigs and fattening pigs in the UK in 1971, it has become an important disease affecting the development of the global pig industry (Zimakov, 1977). Due to the high variability of the PEDV genome, existing commercial vaccines are difficult to effectively control PED. At the same time, the scientific issues about the receptors required for PEDV infection and which host proteins it interacts with are still unclear. Therefore, it is of great significance to find key host factors involved in the PEDV infection process from the host perspective, and provide targets for the development of new antiviral drugs and disease-resistant breeding, which is of great significance for the prevention and control of PEDV. Summary of the invention

[0003] The purpose of the present invention is to provide a preparation for knocking out or inhibiting the expression of the porcine Jagged1 gene for use in resisting porcine epidemic diarrhea virus, wherein the NCBI accession number of the mRNA of the Jagged1 gene in pigs is XM_021077352.1, or the gene encoding the protein shown in SEQ ID NO. 2. In order to achieve the above purpose, the present invention adopts the following technical measures:

[0004] The invention discloses an application of a preparation for knocking out or inhibiting the expression of pig Jagged1 gene in the preparation of an anti-PEDV infection drug; the Jagged1 gene encodes the gene of the protein shown in SEQ ID NO.2.

[0005] In the above-mentioned application, the inhibition method includes inhibition using antisense RNA technology or interfering RNA technology; the knockout method includes: gene knockout using homologous recombination or CRISPR-Cas9 technology; the amount of protein translated by the Jagged1 gene after knockout or inhibition is reduced, has no original function, or cannot be translated into protein, thereby achieving the technical effect of the present invention.

[0006] In the above application, the Jagged1 gene is shown as SEQ ID NO.1.

[0007] When using CRISPR / Cas9 knockout technology, the gRNA is: 5'-GGCCTCCGGTCAGTTCGAGT-3'.

[0008] The protection content of the present invention also includes: preparing a cell model resistant to PEDV infection by knocking out the pig Jagged1 gene.

[0009] Compared with the prior art, the present invention has the following advantages:

[0010] The applicants used CRISPR knockout technology to knock out / inhibit the expression of Jagged1 in porcine cells, and found for the first time that cells with knocked out / inhibited Jagged1 expression can resist PEDV infection by affecting the replication of PEDV. This gene can be used as a new disease resistance target for PEDV. The present invention provides a new target for the development of new drugs that effectively prevent and treat PED, and also provides a new candidate gene for anti-PED breeding research, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 :Construction and verification of plasmid targeting porcine Jagged1 gene

[0012] (A) Design of gRNA targeting porcine Jagged1 gene;

[0013] (B) Bacterial liquid PCR confirmed that the plasmid was successfully constructed;

[0014] (C) Sequencing confirmed that the plasmid was successfully constructed.

[0015] Figure 2 :Construction and verification of LLC-PK1-Cas9-Jagged1 monoclonal cell line

[0016] (A) Genomic DNA sequencing to detect the Jagged1 gene sequence in LLC-PK1-Cas9-Jagged1 cells;

[0017] (B) Western Blot detection of Jagged1 protein expression in LLC-PK1-Cas9-Jagged1 cells;

[0018] (C) MTT assay was used to detect the activity of KO-NC cells and KO-Jagged1 cells.

[0019] Figure 3 :IFA detection of the effect of Jagged1 knockout on PEDV infection in LLC-PK1 cells

[0020] (A) IFA analysis of the effect of Jagged1 knockout on PEDV infection;

[0021] (B)TCID 50 The effect of Jagged1 knockout on PEDV particle release was examined.

[0022] Figure 4 :Showing that knocking out Jagged1 in ST cells inhibits the formation of syncytia by PEDV

[0023] After Jagged1 was knocked out in ST cells, the formation of syncytia was observed under a microscope 12 hours after infection with PEDV (MOI=1). DETAILED DESCRIPTION

[0024] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0025] Embodiment 1:

[0026] Construction and validation of Jagged1 knockout cell lines

[0027] The NCBI number of the mRNA of the target gene Jagged1 gene is XM_021077352.1, the corresponding CDS sequence is shown in SEQ ID NO.1, and it encodes the protein shown in SEQ ID NO.2.

[0028] 1.1 gRNA design and expression vector construction

[0029] (1) gRNA design: Select the partial sequence of the gene location where the gRNA is required to target, and design the sequence of the knockout gRNA. Figure 1 Middle A; gRNA: 5'-GGCCTCCGGTCAGTTCGAGT-3'.

[0030] (2) After diluting the oligo gRNA synthesized by the company to 100 nM, take 5 μL of each complementary sequence and mix them.

[0031] (3) The mixture was placed on a PCR instrument and annealed to obtain double-stranded gRNA. The procedure was as follows: 95°C, 10 min, 65°C, 40 min, 10°C, 10 min.

[0032] (4) The lenti-sgRNA puro (Addgene, 104990) vector was digested with BsmbI and then the gRNA was connected to the vector using T4 ligase.

[0033] (5) Transform the vector into DH5α, plate the plate, and pick single clones for sequencing verification.

[0034] (6) After the positive colonies were correctly identified by PCR and sequencing, the bacterial culture was expanded and the plasmid was extracted according to the operating instructions of the plasmid extraction kit (DP103) of TIANGEN.

[0035] The results are as follows Figure 1 As shown in B and C, the gRNA targeting the Jagged1 gene was successfully connected to the plasmid, indicating that the gRNA expression vector was successfully constructed.

[0036] 1.2 Construction of Jagged1 knockout monoclonal cell line

[0037] (1) According to the operating instructions of the DNA transfection reagent (114-15) of jetPRIME, auxiliary plasmids psPax2 (Addgene, 12260) and pMD2.G (Addgene, 12259) were used to package lentiviral gRNA.

[0038] (2) LLC-PK1-Cas9 cells (the cells are LLC-PK1 cells (pig kidney cell line) that overexpress Cas9 protein, i.e., a cell line that stably expresses Cas9 protein in pig kidney cells) were inoculated into two wells of a 12-well cell culture plate, one well as an experimental group and the other well as a control group, and cultured with DMEM high-glucose medium containing 10% FBS and 1% dual antibody.

[0039] The preparation method of the LLC-PK1-Cas9 cells is as follows:

[0040] LLC-PK1 cells with good growth status, lentivirus containing Cas9 gene (MOI 0.1) and medium containing 8 μg / mL polybrene were plated in 12-well plates and placed in 37°C, 5% CO 2 Culture in an incubator. After 24 hours of lentiviral infection, replace with new complete medium and continue to culture for 24 hours. After 48 hours of lentiviral infection, add 10 μg / mL blasticide for 7 days, digest and dilute the resistant cells, dilute 1:10 times to 96-well plates for culture, select single clones, identify Cas9 gene expression, and the cells with stable expression of Cas9 gene are LLC-PK1-Cas9 cell lines.

[0041] (3) When the cell confluence reaches 60% to 70%, dilute the lentivirus prepared in step 1 with DMEM high-glucose medium containing 10% FBS and 1% double antibody, and add polybrene with a final concentration of 8 μg / mL and lentivirus containing gRNA targeting gene Jagged1 (MOI 1) to incubate the cells.

[0042] (4) One day after infection, cells were subcultured to 10 cm cell culture dishes and cultured further.

[0043] (5) After culturing for 2 days, culture medium (10% FBS, 1% double-antibody-containing DMEM high-glucose medium) containing 3 μg / mL puromycin was added.

[0044] (6) Fresh culture medium containing puromycin was replaced every day. When all cells in the control group died and cells in the experimental group survived, the cells in the experimental group were inoculated into a 96-well cell plate by multiple dilution to form single cells, and puromycin culture medium was added to a final concentration of 3 μg / mL.

[0045] (7) Continue culturing for about 7 days until the single cells grow into monoclonal cell clusters. Select the cells from the monoclonal cell wells, digest them with trypsin, and place them in a 24-well culture plate to obtain a monoclonal cell line.

[0046] 1.3 Verification of Jagged1 protein expression and cell activity in KO-Jagged1 cell lines

[0047] 1.3.1 Detection of Jagged1 gene DNA editing in KO-Jagged1 monoclonal cells by genomic DNA sequencing

[0048] (1) Extraction of cell genomic DNA

[0049] DNA extraction from Jagged1 knockout monoclonal cell lines and control group LLC-PK1-Cas9 cells (abbreviated as KO-NC cells) was performed according to the operating instructions of TIANGEN's blood / cell / tissue genomic DNA extraction kit (DP304).

[0050] (2) PCR amplified the sequence near the Jagged1 gene knockout site and sent it to the company for sequencing. The amplification used "LA Taq" (RR02MQ) from Takara Biotechnology (Dalian) Co., Ltd., and the primers were: Jagged1(pig)-F: ttggGGCCTCCGGTCAGTTCGAGT and Jagged1(pig)-R: aaacACTCGAACTGACCGGAGGCC.

[0051] The PCR program was set as follows: pre-denaturation at 95°C for 10 min, cycle reaction was denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 1 min, a total of 35 cycles, and final extension at 72°C for 5 min. The PCR product was sent to the company for sequencing.

[0052] The sequencing results are as follows Figure 2As shown in A, it was found that the KO-Jagged1 monoclonal cell line lacked 17 base pairs, resulting in a frameshift mutation in the Jagged1 gene, thereby not producing Jagged1 protein. The amplified sequence of the KO-NC cells (i.e., LLC-PK1-Cas9) before knockout is shown in SEQ ID NO.3; and the amplified sequence in the KO-Jagged1 monoclonal cell line after knockout is shown in SEQ ID NO.4.

[0053] 1.3.2 Detection of Jagged1 protein expression in KO-Jagged1 cells by Western Blot

[0054] (1) Preparation of protein samples

[0055] a) After LLC-PK1-Cas9 cells (control group) and KO-Jagged1 monoclonal cell lines (experimental groups, group 1 and group 2, KO-Jagged1-1 and KO-Jagged1-2, respectively) were grown to confluence, the culture medium was aspirated and the cells were washed 1-2 times with pre-cooled PBS.

[0056] b) Add 200 μL of protein lysis buffer (1 μL of protease inhibitor protease inhibitor cocktail (Sigma, P8340) and phosphorylated protease inhibitor PPEi (Sigma, G2007) to each 6-well plate, lyse on ice, and scrape the cells with a cell scraper.

[0057] c) Pipette the lysate into a 1.5 mL centrifuge tube and pipette repeatedly until it becomes clear.

[0058] d) Add the corresponding volume of 5× SDS-PAGE loading buffer, boil in boiling water for 10 min, and store at -80°C.

[0059] (2) SDS-PAGE electrophoresis

[0060] (3) Western Blot analysis:

[0061] The primary antibodies were Jagged1 rabbit polyclonal antibody (Proteintec, 66890-1-Ig) and β-actin mouse monoclonal antibody (Proteintech, 60004-1-Ig);

[0062] a) Secondary antibody incubation: HRP-conjugated anti-mouse IgG (Proteintech, SA00001-1), HRP-conjugated anti-rabbit IgG (Proteintech, SA00001-2);

[0063] b) Protein detection: ECL luminescence method was used.

[0064] The results are as follows Figure 2 As shown in B, Western Blot detection found that the LLC-PK1-Cas9 cells in the control group expressed Jagged1 protein, but the expression of Jagged1 protein in the KO-Jagged1-1 monoclonal cell line in the experimental group was significantly reduced, and Jagged1 protein was not expressed in the KO-Jagged1-2 monoclonal cell line, indicating that the KO-Jagged1 cell line has been successfully constructed.

[0065] 1.3.3 Detection of KO-Jagged1 monoclonal cell line activity using MTT assay

[0066] (1) The same number and appropriate amount of experimental group cells and control group cells were seeded into 96-well plates, with 3 technical replicates in each group. 5% CO 2 , and continue culturing in a 37°C incubator for 48 hours.

[0067] (2) Take out the 96-well plate, add 20 μL of MTT solution to each well, and continue culturing for 4 h in the dark.

[0068] (3) Aspirate the MTT solution and add 150 μL DMSO to each well. Incubate in the dark for 5-10 minutes.

[0069] (4) Use an ELISA reader to measure the absorbance of each well at 490 nm.

[0070] The results of MTT assay are as follows Figure 2 As shown in C, there is no significant difference in the MTT value of cells before and after Jagged1 knockout, indicating that knocking out the Jagged1 gene has no significant effect on cell activity.

[0071] Embodiment 2:

[0072] Indirect immunofluorescence assay (IFA) was used to detect the inhibitory effect of Jagged1 gene knockout on PEDV infection in LLC-PK1 cells

[0073] The same number and appropriate amount of experimental group cells (KO-Jagged1) and control group cells (KO-NC) were seeded into 6-well plates and 5% CO 2 , 37°C incubator. After 24 hours of culture, discard the culture medium and wash twice with DMEM, add PEDV and 10μg / mL trypsin (MOI=1, YN strain, GenBank accession NO.KM597054.1) and mix well, incubate at 37°C for 1 hour, wash twice with PBS, add 1mL DMEM+10μg / mL trypsin culture medium, continue to culture, and collect samples for IFA after 24 hours.

[0074] (1) IFA experiments identified that Jagged1 knockout in KO-Jagged1 cells affects the proliferation of PEDV

[0075] The results are as follows Figure 3 As shown in the figure, at 24h, the fluorescence and the number of PEDV-infected cells in the experimental group (KO-Jagged1) were significantly lower than those in the control group (KO-NC), indicating that knocking out the Jagged1 gene in LLC-PK1 cells significantly inhibited the proliferation of PEDV.

[0076] Using TCID 50 Detection of the effect of Jagged1 gene deletion in LLC-PK1 cells on inhibiting the release of PEDV particles

[0077] The same number and appropriate amount of experimental group cells (KO-Jagged1) and control group cells (KO-NC) were seeded into 6-well plates and 5% CO 2 , 37℃ incubator. After 24h of culture, discard the culture medium and wash twice with DMEM, add PEDV and 10μg / mL trypsin (MOI=1, YN strain, GenBank accession NO.KM597054.1) and mix well. After incubation at 37℃ for 1h, wash twice with PBS, add 1mL DMEM+10μg / mL trypsin culture medium, and continue to culture. After 24h of infection, TCID 50 Detect PEDV titer, the specific method is as follows:

[0078] (1) The virus was diluted 10-fold in series, starting from 10 -1 to 10 -10 .

[0079] (2) After digesting the LLC-PK1 cells that had grown monolayers, cell growth medium was added to prepare a cell suspension, and then 100 μL of the cell suspension was added to each well of a 96-well plate.

[0080] (3) Add 100 μL of the diluted virus solution to each well of a 96-well culture plate, with 8 wells for each dilution. In addition, add 100 μL of cell growth medium to 16 wells of cells as a blank control.

[0081] (4) Place the 96-well plate at 37°C and 5% CO 2 The cells were cultured in an incubator and observed every day. After 3-5 days, the number of wells showing CPE was counted.

[0082] (5) Calculated according to the Reed-Muench method: distance ratio = (percentage of lesion rates above 50% - 50%) / (percentage of lesion rates above - percentage of lesion rates below), lgTCID 50= distance from this case × difference between the logarithms of dilution + the logarithm of the dilution with a lesion rate higher than 50%.

[0083] The results are as follows Figure 3 As shown, after Jagged1 was knocked out in LLC-PK1 cells, the viral titer of PEDV-infected cells decreased by about 10 times, which also indicated that knocking out the expression of this gene in LLC-PK1 cells could inhibit the release of PEDV.

[0084] Embodiment 3:

[0085] IFA analysis was used to detect that knocking out the Jagged1 gene in ST cells inhibited the formation of syncytia by PEDV

[0086] Subsequently, the molecular mechanism of host protein Jagged1 involved in PEDV infection was further explored. The same number of experimental group cells (ST-KO-Jagged1, i.e., ST cells with Jagged1 gene knocked out according to the above steps) and control group cells (ST) were inoculated into 24-well plates and incubated at 5% CO. 2 After culturing at 37°C for 24 h, the culture medium was discarded and the cells were washed twice with DMEM. PEDV and 10 μg / mL trypsin (MOI=1, YN strain, GenBank accession NO.KM597054.1) were added and mixed. After incubation at 37°C for 1 h, the cells were washed twice with PBS. Then 1 mL of DMEM+10 μg / mL trypsin culture medium was added. The cells were cultured for another 12 h and the formation of syncytia was observed.

[0087] (1) The experimental results showed that 12h after PEDV infection, the formation of syncytia in Jagged1 knockout cells was significantly reduced compared with wild-type ST cells (i.e., pig testicular cells). In order to verify the results of (1), the above samples were subjected to indirect immunofluorescence detection.

[0088] (2) Fluorescence observation results are as follows Figure 4 The red arrows indicate that in the wild-type cell group, multiple syncytia can be observed under the natural light of the microscope, and the presence and spread of the virus are also detected at the corresponding positions. However, in the Jagged1 knockout cell group, the formation of syncytia is greatly inhibited, and no obvious syncytia formation is observed. Therefore, it is speculated that during the process of PEDV infection of host cells, the knockout of Jagged1 affects the formation of syncytia and thus inhibits the infection and spread of PEDV.

Claims

1. Use of a preparation for knocking out or inhibiting the expression of porcine Jagged1 gene in the preparation of a drug for resisting porcine epidemic diarrhea virus infection, wherein the Jagged1 gene is a gene encoding the protein shown in SEQ ID NO.

2.

2. The use according to claim 1, wherein the knockout method comprises: Homologous recombination or CRISPR-Cas9 technology, the protein translated from the knocked-out gene has no original function or cannot be translated into protein.

3. The use according to claim 1, wherein the method of suppressing comprises: Antisense RNA technology or interfering RNA technology.

4. The use according to claim 1, wherein the CDS of the Jagged1 gene is shown as SEQ ID NO.

1.

5. The use according to claim 2, wherein the preparation for knocking out the Jagged1 gene in pigs is gRNA: 5'-GGCCTCCGGTCAGTTCGAGT-3'.

6. A cell model for resistance to PEDV infection, characterized in that: The cell model includes cells in which the Jagged1 gene in pig cells is knocked out, the Jagged1 gene encodes the gene of the protein shown in SEQ ID NO.2, the knockout is CRISPR / Cas9 knockout technology, and the gRNA is: 5'-GGCCTCCGGTCAGTTCGAGT-3'.

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