A porcine epidemic diarrhea virus with low replication ability and its construction method and application

Abstract

The present invention discloses a porcine epidemic diarrhea virus with low replication ability, a construction method and an application thereof, and relates to the field of biomedicine technology. The construction method includes the steps of mutating the amino acids 1347 and 1348 of the S protein of porcine epidemic diarrhea virus AH2012 / 12 into serine to construct a recombinant virus; the recombinant virus is a porcine epidemic diarrhea virus with low replication ability. The present invention evaluates the immunogenicity of the recombinant virus, and the results show that the recombinant virus can induce the production of high levels of PEDV S protein IgG antibodies and neutralizing antibodies, and the antibody level produced exceeds that of its parent strain AH2012 / 12 28 days after immunization. These results indicate that the two palmitoylation modification sites of the recombinant virus can increase the antibody level and are important targets for reducing the viral replication ability. The present invention lays an important foundation for the research and development of new attenuated vaccines for porcine epidemic diarrhea virus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a porcine epidemic diarrhea virus with low replication ability, a construction method thereof, and an application thereof. Background Art

[0002] Porcine Epidemic Diarrhea (PED) is a highly contagious enteric disease caused by Porcine epidemic diarrhea virus (PEDV). It is characterized by acute watery diarrhea, vomiting and a high mortality rate of 80%-100% in piglets.

[0003] The S protein of PEDV is a key target for viral invasion and the induction of neutralizing antibodies. Its S1 subunit is responsible for binding to porcine intestinal epithelial cell receptors, while the S2 subunit mediates membrane fusion. Studies have shown that the S protein is a core target for inducing neutralizing antibodies, with antibody levels positively correlated with immune protection. Therefore, the development of vaccines based on the S protein has become a research priority. Currently available vaccines (such as inactivated vaccines or traditional attenuated strains) pose risks of insufficient immunogenicity or reversion to virulence. Therefore, the development of new attenuated PEDV strains is crucial.

[0004] In recent years, reverse genetics has been used to construct recombinant PEDV viruses, achieving controllable attenuation through targeted modification of virulence-related genes. However, existing technologies still face several challenges: First, excessive attenuation (e.g., deletion of the entire S protein domain) can lead to a loss of viral replication and inability to effectively activate humoral immunity; while some mutations can maintain immunogenicity, their attenuation effects are unstable; and second, most studies focus on glycosylation modifications of the S protein, with limited research on other modifications.

[0005] Proteins acquire physiological functions through multiple complex processes, including gene transcription, post-transcriptional processing, translation, post-translational modification, and transport. Post-translational modification (PTM) refers to a covalent modification catalyzed by specific enzymes after protein synthesis, adding functional groups to one or more amino acid residues in the form of covalent bonds. These modifications play a crucial role in regulating protein solubility, activity, stability, subcellular localization, and mediating protein interactions. Common PTMs include phosphorylation, glycosylation, methylation, hydroxylation, ubiquitination, and lipidation. Lipidation modifications include N-myristoylation, palmitoylation, and farnesylation. Palmitoylation can be categorized by attachment method: N-palmitoylation, O-palmitoylation, and S-palmitoylation. The first two types are irreversible, while S-palmitoylation is the only reversible lipidation modification. Therefore, S-palmitoylation is the primary palmitoylation reported in the literature. S-palmitoylation modification refers to the covalent modification of long-chain fatty acids (usually 16-carbon palmitic acid) to protein cysteine ​​residues through thioester bonds. It can be dynamically regulated by catalysis and inhibition by palmitoyltransferase and palmitoyl protein thioesterase, respectively, thereby affecting protein hydrophobicity, subcellular localization, transport, stability, etc.

[0006] Studies have shown that severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and mouse hepatitis coronavirus (MHV) all contain palmitoylated proteins. CEMcBride et al. mutated nine cysteine ​​residues on the SARS-CoV S protein that could potentially be palmitoylated and found that the S protein's stability, localization, transport, and interaction with the M protein were unaffected, but the virus's membrane fusion function was inhibited. Lisa A. Lopez et al. confirmed palmitoylation of the MHV E protein at position A59, and JA Boscarino et al. further found that palmitoylation of the MHV E protein is crucial for viral assembly. To date, the palmitoylation modification of SARS-CoV-2 has been the most extensively studied: studies have demonstrated that the SARS-CoV-2 S protein is palmitoylated at both C15 and the cytoplasmic tail, and that this is crucial for S-mediated syncytium formation and SARS-CoV-2 pseudovirion entry; studies have found that 2-BP can reduce SARS-CoV-2 membrane fusion ability and viral infectivity; and studies have found that palmitoylation of the E protein enhances the electrostatic forces on both sides of the pentamer to attract and release cations in the cavity to the cell membrane. However, studies on the palmitoylation of PEDV have not yet been reported. Summary of the Invention

[0007] The present invention aims to provide a low-replication porcine epidemic diarrhea virus (PEDV), its construction method, and its application, to address the aforementioned problems of the prior art. Two palmitoylation sites in this low-replication porcine epidemic diarrhea virus (PEDV) enhance antibody levels and serve as important targets for attenuating viral replication. This invention lays an important foundation for the development of new attenuated PEDV vaccines.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for constructing a porcine epidemic diarrhea virus with low replication ability, comprising the steps of mutating the amino acids 1347 and 1348 of the S protein of porcine epidemic diarrhea virus AH2012 / 12 into serine to construct a recombinant virus; the recombinant virus is the porcine epidemic diarrhea virus with low replication ability.

[0010] Furthermore, the CRISPR / Cas9 gene editing method was used to mutate the amino acids at positions 1347 and 1348 into serine.

[0011] Furthermore, the CRISPR / Cas9 gene editing method comprises the following steps:

[0012] Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, primers C1347+1348S-F and C1347+1348S-R were used to amplify fragment 1, and primers C1347+1348S-MF and C1347+1348S-MR were used to amplify fragment 2;

[0013] Using the fragment 1 and the fragment 2 as templates, primers C1347+1348S-F and C1347+1348S-MR were used to amplify the target fragment;

[0014] Using sgRNA-F and sgRNA-R, the pBAC-AH2012 / 12 infectious clone plasmid was cleaved in vitro by CRISPR / Cas9 to obtain enzyme digestion products;

[0015] Cloning the target fragment into the enzyme-digested product by homologous recombination to obtain a recombinant plasmid;

[0016] transfecting the recombinant plasmid into a host cell to obtain the porcine epidemic diarrhea virus with low replication ability;

[0017] The nucleotide sequences of the sgRNA-F and the sgRNA-R are shown in SEQ ID NO. 1-2, respectively;

[0018] The nucleotide sequences of the C1347+1348S-F, the C1347+1348S-R, the C1347+1348S-MF and the C1347+1348S-MR are shown in SEQ ID NOs. 4-7, respectively.

[0019] Furthermore, the CRISPR / Cas9 in vitro cleavage reaction system is: 5 μL of 10×NE buffer, 5 μL of Cas9 enzyme, 10 μL each of sgRNA-F and sgRNA-R, 2-5 μg of pBAC-AH2012 / 12 infectious clone plasmid, and sterile enzyme-free water supplemented to 50 μL; and / or

[0020] The reaction conditions for the CRISPR / Cas9 in vitro cleavage were incubation at 37°C for 2.5 h.

[0021] Furthermore, the homologous recombination reaction system is: 5 μL of 2× seamless cloning recombinase mixture, 2 μL of target fragment and 3 μL of enzyme digestion product; and / or

[0022] The reaction conditions for the homologous recombination are incubation at 50° C. for 15-30 min.

[0023] Furthermore, the host cell is an African green monkey kidney cell.

[0024] The present invention also provides a porcine epidemic diarrhea virus with low replication ability constructed according to the above construction method.

[0025] The present invention also provides the use of the porcine epidemic diarrhea virus with low replication ability in the preparation of an attenuated porcine epidemic diarrhea vaccine.

[0026] The present invention also provides an attenuated porcine epidemic diarrhea vaccine, the active ingredient of which includes the porcine epidemic diarrhea virus with low replication ability.

[0027] Furthermore, the porcine epidemic diarrhea attenuated vaccine also includes a vaccine adjuvant.

[0028] The present invention discloses the following technical effects:

[0029] The present invention targeted two sgRNAs to cut and homologously recombine the AH2012 / 12 full-gene BAC recombinant plasmid, mutating the cysteine ​​residues 1347 and 1348 of the S protein, which are susceptible to palmitoylation, to serine. This recombinant BAC plasmid was then transfected into Vero cells, resulting in the rescue of the recombinant virus r12-C1347+1348S, which harbors mutations at the palmitoylation sites of the S protein. Viral gene sequence analysis, indirect immunofluorescence assays, plaque assays, and growth curve analysis confirmed the successful rescue of the recombinant virus strain. Mouse experiments evaluated the immunogenicity of the recombinant virus, demonstrating that the recombinant virus strain r12-C1347+1348S induced high levels of PEDV S protein IgG and neutralizing antibodies in mice, with the antibody levels exceeding those of its parental strain AH2012 / 12 28 days after immunization. These results indicate that the two palmitoylation sites of the r12-C1347+1348S mutation can increase antibody levels in mouse serum and are important targets for reducing viral replication. This invention lays an important foundation for the development of new attenuated PEDV vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 The electrophoresis detection diagram of the target fragment; A is the electrophoresis detection diagram of amplified fragment 1 and amplified fragment 2; B is the electrophoresis detection diagram of the final target fragment; in A, M: DL2000 Marker; 1: amplified fragment 1; 2: amplified fragment 2; in B, M: DL2000 Marker; 1: final target fragment;

[0032] Figure 2 This is the electrophoresis detection diagram of the CRISPR / Cas9 in vitro cleavage of the pBAC-AH2012 / 12 plasmid; M: 1Kb Marker; 1: cleaved pBAC-AH2012 / 12 plasmid; 2: uncut pBAC-AH2012 / 12 plasmid;

[0033] Figure 3 The figure shows the PCR identification results of the bacterial solution after homologous recombination product transformation; where: M: DL2000 Marker; 1-5: bands of 5 single colonies identified by PCR using PEDV-F and PEDV-R primers;

[0034] Figure 4This is the observation picture of syncytial lesions after recombinant plasmid transfection into Vero cells;

[0035] Figure 5 This is the result of gene sequencing;

[0036] Figure 6 The results of indirect immunofluorescence identification are shown; MOCK represents the negative control group of empty transfection;

[0037] Figure 7 This is the result of the plaque growth test;

[0038] Figure 8 This is the growth curve measurement result diagram;

[0039] Figure 9 The results of the test of IgG antibody level in mouse serum; * P < 0.05; ** P<0.01. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1

[0046] 1. Materials and Methods

[0047] 1.1 Main Materials

[0048] The porcine epidemic diarrhea virus (PEDV) AH2012 / 12 strain, PEDV N monoclonal antibody (PEDV N mAb), and African green monkey kidney (Vero) cells were provided by the Veterinary Institute of Jiangsu Academy of Agricultural Sciences. The BAC plasmid vector is maintained in our laboratory. The pBAC-AH2012 / 12 infectious clone plasmid, a full-genome BAC recombinant plasmid of the AH2012 / 12 strain, was provided by the Veterinary Institute of Jiangsu Academy of Agricultural Sciences and has been published in the literature "Yin Liping, Chang Changlin, Fan Baochao, et al. Construction of a chimeric strain of the S gene of a porcine epidemic diarrhea virus variant strain and an S-indel epidemic strain [J / OL]. Chinese Journal of Veterinary Science, 1-8 [2025-04-30]." NEB 10-beta competent cells were purchased from Beijing Biomade Gene Technology Co., Ltd. 0.25% trypsin, fetal bovine serum (FBS), MEM (2×), and DMEM cell culture media were purchased from Gibco; reverse transcriptase, 2× high-fidelity DNA polymerase (Phanta Max MasterMix), and a total RNA extraction kit were purchased from Vazyme; 2× seamless cloning recombinase mix (SeamLess CloningMix) was purchased from Beijing Biomed Gene Technology Co., Ltd.; DNA purification kits and gel extraction kits were provided by Guangzhou Omega; the Large Construct Kit was purchased from QIAGEN; Cas9 nuclease (S. Pyogenes) and T7 RNA polymerase were purchased from New England Biolabs (Beijing). Transfection reagent Lipofectamine™ 3000, yeast extract, and tryptone were purchased from Thermo Fisher Scientific; DAPI staining solution and horseradish peroxidase-conjugated goat anti-mouse IgG antibody were purchased from Beyotime; and methylcellulose was purchased from Sigma. Fifteen 6-week-old male BALB / c mice were purchased from the Animal Experimental Center of Yangzhou University. HRP-labeled goat anti-mouse IgG was purchased from abcam; one-component TMB colorimetric solution was purchased from Huzhou Yingchuang Biotechnology Co., Ltd.; sulfuric acid was purchased from Kelon Chemical Reagent Factory; and Tween 20 was purchased from Solebol.

[0049] 1.2 Primer sequences

[0050] Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, specific guide RNAs (sgRNAs) were designed to target the S region of the PEDV gene. sgRNA-F and sgRNA-R were amplified using scaffold oligonucleotides (scafold-oligo) to obtain double-stranded DNA versions. sgRNA-F and sgRNA-R were designed and then amplified using PCR to obtain double-stranded DNA versions of sgRNA-F and sgRNA-R, respectively. The scafold-oligo provided the scaffold sequence for sgRNA-F and sgRNA-R. The PCR system was as follows: 3 μL of sgRNA-F (or sgRNA-R), 3 μL of scafold-oligo, 25 μL of 2× high-fidelity enzyme, and 19 μL of water. The PCR program was pre-denaturation at 95°C for 3 minutes, followed by 35 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 1 minute, followed by 72°C for 5 minutes and 4°C. Double-stranded DNA of approximately 100 bp was generated. Amplified products were purified using a DNA purification kit (Omegea Bio-Tek, Guangzhou, China) according to the manufacturer's instructions.

[0051] Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, a pair of specific primers, C1347+1348S-F and C1347+1348S-R, were designed to amplify fragment 1. Specific primers, C1347+1348S-MF and C1347+1348S-MR, were designed to amplify fragment 2. Finally, using fragments 1 and 2 as templates, C1347+1348S-F and C1347+1348S-MR were used to amplify the final target fragment. Finally, a pair of identification primers was designed to verify successful mutation of the strain. Primer sequences are shown in Table 1.

[0052] Table 1 Primer sequences

[0053]

[0054] 1.3 Amplification of target fragments containing mutations

[0055] Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, specific primers C1347+1348S-F and C1347+1348S-R were used to amplify fragment 1. Specific primers C1347+1348S-MF and C1347+1348S-MR were used to amplify fragment 2. Finally, using fragments 1 and 2 as templates, specific primers C1347+1348S-F and C1347+1348S-MR were used to amplify the final target fragment. The PCR program was as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 1 min, followed by 72°C for 5 min and 4°C. The PCR system consisted of 2 μL of each primer, 2 μL of template, 25 μL of 2× Phanta Max Master Mix High-Fidelity Enzyme, and 19 μL of water.

[0056] 1.4 CRISPR / Cas9 in vitro cleavage of the pBAC-AH2012 / 12 plasmid

[0057] The double-stranded DNA product of the sgRNA obtained in part 1.2 was transcribed in vitro and purified to obtain the RNA product of the sgRNA.

[0058] The pBAC-AH2012 / 12 infectious clone plasmid was excised in vitro using Cas9 nuclease. PCR products were detected by 0.8% agarose gel electrophoresis. The reaction system was as follows: 5 μL of NE Buffer r3.1 (10×); 5 μL of Cas9 enzyme; 10 μL each of sgRNA-F and sgRNA-R; 2 μg of pBAC-AH2012 / 12 infectious clone plasmid (up to 5 μg); and sterile, enzyme-free water to make up to 50 μL. The reaction was incubated at 37°C for 2.5 hours.

[0059] 1.5 Homologous recombination and ligation transformation

[0060] Using homologous recombinase, ligate the DNA fragment containing the single-point mutation (i.e., the final target fragment amplified in Section 1.3) with the linearized, purified BAC plasmid (i.e., the pBAC-AH2012 / 12 plasmid digested in Section 1.4). The reaction mixture consists of 5 μL of 2× Seamless Cloning Recombinase Mix, 2 μL of the DNA fragment, and 3 μL of the digested product. Incubate at 50°C for 20 min (a 15-30 min incubation will achieve equivalent results). After ligation, store in a -20°C freezer until needed. Place NEB10-beta competent cells in an ice-water mixture and allow them to slowly thaw. Under a laminar flow hood, add the entire ligation product to the competent cells. Then, gently tap the centrifuge tube to mix thoroughly and place it in ice-water for 30 min. After incubation, quickly transfer the competent cells to a water bath set at 42°C and heat shock them for 50 s. After re-incubating in an ice-water bath for 2 minutes, add 600 μL of antibiotic-free liquid LB medium and shake at 37°C, 200 rpm, for 1 hour. Centrifuge at 5000 rpm for 1 minute, discard 500 μL of LB medium, resuspend the bacterial mass in the remaining liquid, and spread the liquid onto agar plates. Allow the liquid to completely absorb the plate, then invert and incubate in a 37°C incubator overnight.

[0061] 1.6 PCR identification of bacterial culture and extraction of recombinant plasmid

[0062] Single colonies were picked from the plates and placed in LB medium supplemented with the appropriate antibiotics. The culture was shaken at 37°C and 200 rpm. Once the culture became turbid, PCR was performed using primers PEDV-F and PEDV-R. PCR products showing bands were sent to Sangon for sequencing. Single colonies identified by sequencing were selected for amplification and plasmid extraction using the QIAGEN Large Construct kit instructions. The concentration was determined and the cells were stored at -80°C.

[0063] 1.7 Transfection rescue of BAC recombinant plasmid containing mutation

[0064] Vero cells were plated in six-well plates one day in advance and transfected with 6 μg of the recombinant plasmid extracted from fraction 1.6 after reaching 70% confluence. After transfection, the six-well plates were placed in a 37°C, 5% CO2 incubator for further culture. When cells became pathological, they were frozen and thawed three times and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected to obtain the recombinant virus, a low-replication-competent porcine epidemic diarrhea virus (designated r12-C1347+1348S), and stored at -80°C.

[0065] 1.8 Indirect immunofluorescence assay

[0066] 24-well plates filled with Vero cells were infected with the recombinant virus at an MOI of 0.01, and the supernatant was discarded after 24 hours. The cells were fixed with fixative (methanol:acetone = 1:4) for 15 minutes, and the solution was discarded. After washing with PBS, 500 μL of mouse anti-PEDV N mAb (1:500 dilution) was added to each well, and the cells were incubated at 37°C for 1.5 hours, and the solution was discarded. After washing with PBS, 500 μL of FITC-conjugated goat anti-mouse IgG (1:2000 dilution) was added to each well, and the cells were incubated at 37°C in the dark for 1 hour. After washing with PBS three times, the cells were observed under a fluorescence microscope for determination.

[0067] 1.9 Plaque assay

[0068] The culture medium of the Vero cells grown in the six-well plate was discarded, and the Vero cells were washed twice with serum-free DMEM. The recombinant virus was diluted 10-fold, starting from 10 -1 Until 10 -5 500 μL of virus solution at different dilutions was used to infect different wells of Vero cells. After incubation at 37°C for 1.5 hours, the virus solution was discarded and the Vero cells were washed twice with DMEM. Finally, 2 mL of plaque fixative solution was added to cover the Vero cells. Incubate at 37°C for 24-48 hours and observe the cells daily for the appearance of plaques. If plaques appear, the plaque solution was discarded, and the cells were washed twice with DMEM before adding 1.5 mL of 0.1% crystal violet. Stain the cells in the dark at 37°C for 1-2 hours, discard the crystal violet dye, and observe the plaque morphology.

[0069] 1.10 virus half-cell infectious dose (TCID 50 ) determination

[0070] Take 100 μL of virus solution, add 900 μL of DMEM maintenance solution, and dilute 10-fold to 10 -7 100 μL was inoculated into 96-well plates of well-grown Vero cells, with eight replicates. After 1.5 h of incubation, the solution was discarded and 500 μL of DMEM maintenance medium was added. Pathological changes were observed within 72 h. TCID was calculated using the Reed-Muench method. 50 .

[0071] 1.11 Growth curve identification

[0072] A 24-well plate of Vero cells was infected with the parental virus and 10 recombinant viruses at an MOI of 0.01. Viruses were collected at four time points, 6 h, 12 h, 24 h, and 36 h after infection, and three replicate wells were set up. Incubate in a 37°C incubator for 1.5 h, discard the supernatant of the virus solution, wash the Vero cells twice with DMEN, add 500 μL of virus maintenance solution to each well of the 24-well plate, and continue to incubate in a 37°C incubator. Collect the cell supernatant at 6 h, 12 h, 24 h, and 36 h after infection, and determine its TCID 50 According to the measured virus TCID 50 Draw the growth curve of each virus strain.

[0073] 1.12 Design of recombinant virus mouse experiment

[0074] The recombinant virus strain r12-C1347+1348S and the parental strain AH2012 / 12 were inoculated into 6-week-old BALB / c mice, and a negative control group inoculated with DMEM was established to investigate the immunogenicity of viruses with mutated PEDV S protein palmitoylation sites, providing important clues for understanding the pathogenicity of PEDV and developing new vaccines. The mouse experimental design is shown in Table 2. Both the parental strain AH2012 / 12 and the recombinant virus r12-C1347+1348S were diluted to 1×10 5.50 TCID 50 / mL. On the day of immunization, the mixture was mixed with adjuvant ISA206 and injected intramuscularly with 300 μL of DMEM per mouse. The MOCK group received an intramuscular injection of 300 μL of DMEM. Blood was collected before the first immunization to assess serum antibody levels. Day 0 was designated as the first immunization. Blood was collected 14 days after the first immunization for the second immunization, 28 days after the first immunization for the third immunization, 42 days after the first immunization for the fourth immunization, and 56 days after the first immunization for the final blood collection and sacrifice. Antibody levels in mouse serum were measured using ELISA.

[0075] Table 2 Mouse experiment design

[0076]

[0077] 2. Results

[0078] 2.1 Amplification of target fragments containing mutations

[0079] Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, specific primers C1347+1348S-F and C1347+1348S-R were used to amplify fragment 1, and specific primers C1347+1348S-MF and C1347+1348S-MR were used to amplify fragment 2. Finally, using fragments 1 and 2 as templates, specific primers C1347+1348S-F and C1347+1348S-MR were used to amplify the final target fragment. The final target fragment was approximately 1000 bp long ( Figure 1 ).

[0080] 2.2 CRISPR / Cas9 in vitro cleavage of the pBAC-AH2012 / 12 plasmid

[0081] The pBAC-AH2012 / 12 infectious clone plasmid was cut in vitro using NEB's Cas9 nuclease. Figure 2 ) showed that a band of approximately 1000 bp was cut out.

[0082] 2.3 PCR identification of homologous recombination products after transformation

[0083] The amplified product containing the mutation was cloned into the cut pBAC-AH2012 / 12 linear plasmid by homologous recombination and then transformed into competent NEB 10-beta cells. After culture, the cells were evenly plated on LB plates containing 25 μg / mL chloramphenicol for screening. Five single colonies were selected for PCR identification using PEDV-F and PEDV-R primers. Electrophoresis results ( Figure 3 ) showed that 5 colonies had brighter bands around 1000 bp, which was consistent with expectations.

[0084] 2.4 Rescue, sequence analysis, and IFA identification of chimeric recombinant viruses

[0085] The recombinant plasmid was transfected into Vero cells, and syncytial lesions appeared in the cells 36 h later ( Figure 4 ), indicating that the rescue was successful. After extracting RNA and reverse-transcribing cDNA, PCR amplification was performed using identification primers PEDV-F and PEDV-R. The amplified product was sent to Shanghai Sangon Biotechnology for sequencing. The results were ( Figure 5 ) showed that the sequence was correct, and the 1347th and 1348th cysteine ​​residues were successfully mutated to serine residues. The results of the indirect immunofluorescence test confirmed that the virus was PEDV ( Figure 6 ).

[0086] 2.5 Comparison of biological characteristics of recombinant viruses

[0087] Plaque assay results ( Figure 7) showed that the ability of the recombinant virus r12-C1347+1348S to form plaques was relatively weaker than that of the parent virus AH2012 / 12, and the plaque area was smaller. Growth curve test results ( Figure 8 ) showed that the virus titer of the recombinant virus r12-C1347+1348S was significantly lower than that of the parental virus AH2012 / 12, with a decrease of about 2 titers at 12 hpi.

[0088] 2.6 Detection of mouse serum antibody levels

[0089] Blood was collected from the tail vein on days 14, 28, 42, and 56 after vaccination, and serum samples were prepared. Porcine epidemic diarrhea virus spike protein (PEDV S) was used to coat 96-well plates, and the dynamic changes in S protein-specific immunoglobulin G (IgG) antibodies in the serum of mice in the experimental groups were quantitatively detected by indirect enzyme-linked immunosorbent assay (ELISA). Figure 9 ) showed that both the parental virus AH2012 / 12 and the recombinant virus r12-C1347+1348S induced high levels of S protein antibodies compared with the negative control group, and the recombinant virus r12-C1347+1348S induced antibody levels exceeding that of the parental virus rAH2012 / 12 starting 42 days after immunization.

[0090] In summary, based on the AH2012 / 12 infectious cloning platform, the present invention successfully constructed a recombinant virus with two mutations, r12-C1347+1348S, using CRISPR / Cas9-mediated genome editing technology. The immunogenicity of this recombinant virus was evaluated in mice. The results showed that the recombinant virus r12-C1347+1348S induced high levels of IgG and neutralizing antibodies against the PEDV S protein in mice, with antibody levels exceeding those of the parental strain AH2012 / 12 28 days after immunization. These results suggest that the two palmitoylation sites of the r12-C1347+1348S mutation can elevate antibody levels in mouse serum, making it an important target for attenuating viral replication.

[0091] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing a porcine epidemic diarrhea virus with low replication ability, characterized in that: The method comprises the steps of mutating the amino acids 1347 and 1348 of the S protein of porcine epidemic diarrhea virus AH2012 / 12 into serine to construct a recombinant virus; the recombinant virus is the porcine epidemic diarrhea virus with low replication ability.

2. The construction method according to claim 1, characterized in that The CRISPR / Cas9 gene editing method was used to mutate the amino acids at positions 1347 and 1348 into serine.

3. The construction method according to claim 2, characterized in that The CRISPR / Cas9 gene editing method comprises the following steps: Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, primers C1347+1348S-F and C1347+1348S-R were used to amplify fragment 1, and primers C1347+1348S-MF and C1347+1348S-MR were used to amplify fragment 2; Using the fragment 1 and the fragment 2 as templates, primers C1347+1348S-F and C1347+1348S-MR were used to amplify the target fragment; Using sgRNA-F and sgRNA-R, the pBAC-AH2012 / 12 infectious clone plasmid was cleaved in vitro by CRISPR / Cas9 to obtain enzyme digestion products; Cloning the target fragment into the enzyme-digested product by homologous recombination to obtain a recombinant plasmid; transfecting the recombinant plasmid into a host cell to obtain the porcine epidemic diarrhea virus with low replication ability; The nucleotide sequences of the sgRNA-F and the sgRNA-R are shown in SEQ ID NO. 1-2, respectively; The nucleotide sequences of the C1347+1348S-F, the C1347+1348S-R, the C1347+1348S-MF and the C1347+1348S-MR are shown in SEQ ID NOs. 4-7, respectively.

4. The construction method according to claim 3, characterized in that The CRISPR / Cas9 in vitro cleavage reaction system is as follows: 5 μL of 10×NE buffer, 5 μL of Cas9 enzyme, 10 μL each of sgRNA-F and sgRNA-R, 2-5 μg of pBAC-AH2012 / 12 infectious clone plasmid, and sterile enzyme-free water to 50 μL; and / or The reaction conditions for the CRISPR / Cas9 in vitro cleavage were incubation at 37°C for 2.5 h.

5. The construction method according to claim 3, characterized in that The homologous recombination reaction system is: 5 μL of 2× seamless cloning recombinase mixture, 2 μL of target fragment and 3 μL of enzyme digestion product; and / or The reaction conditions for the homologous recombination are incubation at 50° C. for 15-30 min.

6. The construction method according to claim 3, characterized in that: The host cell is an African green monkey kidney cell.

7. A porcine epidemic diarrhea virus with low replication ability constructed according to the construction method according to any one of claims 1 to 6.

8. Use of the porcine epidemic diarrhea virus with low replication capacity as claimed in claim 7 in the preparation of an attenuated porcine epidemic diarrhea vaccine.

9. An attenuated porcine epidemic diarrhea vaccine, characterized in that: The active ingredient comprises the porcine epidemic diarrhea virus with low replication ability as claimed in claim 7.

10. The attenuated porcine epidemic diarrhea vaccine according to claim 9, characterized in that The attenuated porcine epidemic diarrhea vaccine also includes a vaccine adjuvant.

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