Porcine epidemic diarrhea virus with low replication capability and construction method and application thereof

Abstract

The invention discloses a porcine epidemic diarrhea virus with low replication capacity as well as a construction method and application thereof, and relates to the technical field of biological medicines. The construction method comprises the following steps: mutating 1347th and 1348th amino acids of S protein of the porcine epidemic diarrhea virus AH2012 / 12 into serine, and constructing to obtain a recombinant virus; the recombinant virus is the porcine epidemic diarrhea virus with low replication capability. By evaluating the immunogenicity of the recombinant virus, the result shows that the recombinant virus can induce generation of high-level PEDV S protein IgG antibodies and neutralizing antibodies, and the antibody generation level exceeds that of a parent strain AH2012 / 12 after immunization for 28 days. The results show that the two palmitoylation modification sites of the recombinant virus can increase the antibody level and are important targets for weakening the virus replication ability. The invention lays an important foundation for research and development of novel attenuated vaccines of the porcine epidemic diarrhea virus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly 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 intestinal disease caused by Porcine epidemic diarrhea virus (PEDV), which is characterized by acute watery diarrhea, vomiting in piglets and a high mortality rate of up to 80%-100%.

[0003] The S protein of PEDV is a key target for mediating virus invasion and inducing neutralizing antibodies. Its S1 subunit is responsible for binding to the receptor of porcine intestinal epithelial cells, and the S2 subunit mediates membrane fusion. Research shows that the S protein is the core target for inducing neutralizing antibodies, and the level of its antibodies is positively correlated with the immune protection effect. Therefore, the development of vaccines based on the S protein has become the research focus. Currently, commercially available vaccines (such as inactivated vaccines or traditional attenuated strains) have problems of insufficient immunogenicity or the risk of virulence reversion. Based on this, it is very necessary to develop new PEDV attenuated strains.

[0004] In recent years, reverse genetics technology has been used to construct PEDV recombinant viruses, and controllable attenuation has been achieved by targeting and modifying virulence-related genes. However, the existing technology still faces some problems: one is that excessive attenuation (such as deleting the entire S protein domain) will lead to the loss of virus replication ability and the inability to effectively activate humoral immunity, and although some mutations can maintain immunogenicity, the attenuation effect is unstable; the other is that most studies focus on the glycosylation modification of the S protein, and less research has been done on other modifications.

[0005] Proteins need to go through multiple complex processes before they acquire physiological functions, including gene transcription, post-transcriptional processing, translation, post-translational modification, and transportation, etc. Among them, post-translational modification (PTM) refers to a type of covalent modification catalyzed by specific enzymes after protein synthesis, adding corresponding functional groups to one or more amino acid residues of the protein in the form of covalent bonds, thereby playing an important role in regulating protein solubility, activity, stability, subcellular localization, and mediating protein interactions. Common PTMs include phosphorylation, glycosylation, methylation, hydroxylation, ubiquitination, and lipidation, etc. Lipidation modification includes N-myristoylation, palmitoylation, and farnesylation. According to the linkage mode, palmitoylation can be divided into N-palmitoylation, O-palmitoylation, and S-palmitoylation. The first two types are irreversible modifications, while S-palmitoylation is the only reversible lipidation modification. Therefore, the palmitoylation modification reported in the literature is mainly S-palmitoylation. S-palmitoylation modification refers to the covalent modification of long-chain fatty acids (usually palmitic acid with 16 carbons) to protein cysteine residues through thioester bonds, which can be dynamically regulated by palmitoyltransferases and palmitoyl protein thioesterases for catalysis and inhibition respectively, thereby affecting protein hydrophobicity, subcellular localization, transportation, stability, etc.

[0006] Current studies have shown that palmitoylated proteins exist in Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and Mouse Hepatitis Coronavirus (MHV). After CEMcBride et al. mutated 9 cysteine residues on the SARS-CoV S protein that might be palmitoylated, they found that the stability, localization, transportation, and interaction with the M protein of the S protein were not affected, but the membrane fusion function of the virus was inhibited. Lisa A Lopez et al. confirmed that the MHV E protein was palmitoylated at the A59 site, and J.A. Boscarino et al. further found that the palmitoylation of the MHV E protein was crucial for virus assembly. So far, the palmitoylation modification of SARS-CoV-2 has been the most extensively studied: Some studies have proved that the S protein of SARS-CoV-2 is palmitoylated at C15 and at the cytoplasmic tail, and it is crucial for S-mediated syncytium formation and the entry of SARS-CoV-2 pseudovirus particles; Some studies have found that 2-BP can reduce the membrane fusion ability and virus infectivity of SARS-CoV-2. Some studies have found that after the E protein is palmitoylated, the electrostatic force on both sides of the pentamer is more conducive to attracting and releasing cations in the cavity to the cell membrane. However, there has been no report on the palmitoylation study of PEDV. Summary of the Invention

[0007] The object of the present invention is to provide a porcine epidemic diarrhea virus with low replication ability, its construction method and application, so as to solve the problems existing in the above-mentioned prior art. The two palmitoylation modification sites of the porcine epidemic diarrhea virus with low replication ability can increase the antibody level and are important targets for weakening the virus replication ability. The present invention lays an important foundation for the research and development of a new attenuated vaccine against porcine epidemic diarrhea virus.

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

[0009] The present invention provides a construction method of a porcine epidemic diarrhea virus with low replication ability, including the step of mutating the 1347th and 1348th amino acids 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] Further, the CRISPR / Cas9 gene editing method is used to mutate the 1347th and 1348th amino acids into serine.

[0011] Further, the CRISPR / Cas9 gene editing method includes the following steps:

[0012] Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, fragment 1 is amplified with primers C1347+1348S-F and C1347+1348S-R, and fragment 2 is amplified with primers C1347+1348S-M-F and C1347+1348S-M-R;

[0013] Using the fragment 1 and the fragment 2 as templates, the target fragment is amplified with primers C1347+1348S-F and C1347+1348S-M-R;

[0014] Using sgRNA-F and sgRNA-R, the pBAC-AH2012 / 12 infectious clone plasmid is subjected to CRISPR / Cas9 in vitro cleavage to obtain a cleavage product;

[0015] Through homologous recombination, the target fragment is cloned into the cleavage product to obtain a recombinant plasmid;

[0016] The recombinant plasmid is transfected 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 respectively shown in SEQ ID NO.1-2;

[0018] The nucleotide sequences of the C1347+1348S-F, C1347+1348S-R, C1347+1348S-M-F, and C1347+1348S-M-R are shown in SEQ ID NO.4-7 respectively.

[0019] Furthermore, the reaction system for in vitro cleavage by CRISPR / Cas9 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 is added to make up to 50 μL; and / or

[0020] The reaction condition for in vitro cleavage by CRISPR / Cas9 is incubation at 37°C for 2.5 h.

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

[0022] The reaction condition for homologous recombination is incubation at 50°C for 15-30 min.

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

[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 application of the above porcine epidemic diarrhea virus with low replication ability in the preparation of a porcine epidemic diarrhea attenuated vaccine.

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

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

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

[0029] In the present invention, two sgRNAs were designed by targeting to cleave the whole-genome BAC recombinant plasmid of AH2012 / 12 and perform homologous recombination, and the cysteines at positions 1347 and 1348 of the S protein that can undergo palmitoylation modification were mutated into serine, obtaining a recombinant BAC plasmid, which was transfected into Vero cells, and a recombinant virus r12-C1347+1348S with mutated palmitoylation modification sites of the S protein was rescued. The rescue of this recombinant virus strain was proved by virus gene sequence analysis, indirect immunofluorescence assay, plaque assay, growth curve determination, etc. The immunogenicity of this recombinant virus was evaluated through mouse experiments, and the results showed that the recombinant virus strain r12-C1347+1348S could induce the production of high levels of PEDV S protein IgG antibodies and neutralizing antibodies in mice, and the antibody levels produced exceeded those of its parental strain AH2012 / 12 28 days after immunization. These results indicate that the two mutated palmitoylation modification sites of r12-C1347+1348S can increase the antibody levels in mouse serum and are important targets for weakening the virus replication ability. The present invention has laid an important foundation for the research and development of new attenuated vaccines against PEDV. Brief Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 Electrophoresis detection map of the target fragment; among them, A is the electrophoresis detection map of amplification fragment 1 and amplification fragment 2; B is the electrophoresis detection map of the final target fragment; in A, M: DL2000 Marker; 1: amplification fragment 1; 2: amplification fragment 2; in B, M: DL2000 Marker; 1: final target fragment;

[0032] Figure 2 Electrophoresis detection map of the CRISPR / Cas9 in vitro cleavage of pBAC-AH2012 / 12 plasmid experiment; among them, M: 1Kb Marker; 1: cleaved pBAC-AH2012 / 12 plasmid; 2: uncleaved pBAC-AH2012 / 12 plasmid;

[0033] Figure 3 PCR identification result map of the bacterial liquid after transformation of the homologous recombination product; among them: M: DL2000 Marker; 1-5: bands of PCR identification of 5 single colonies using PEDV-F and PEDV-R primers;

[0034] Figure 4Observation diagram of syncytial lesions after transfection of recombinant plasmid into Vero cells; among them, MOCK represents the negative control group transfected with empty vector.

[0035] Figure 5 Diagram of gene sequencing results.

[0036] Figure 6 Diagram of indirect immunofluorescence identification results.

[0037] Figure 7 Diagram of the results of plaque growth assay.

[0038] Figure 8 Diagram of the results of growth curve determination.

[0039] Figure 9 Diagram of the detection results of IgG antibody levels in mouse serum; among them, * P < 0.05; ** P < 0.01. Detailed implementation manners

[0040] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0043] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0044] As for the terms "comprising", "including", "having", "containing", etc. used in this article, they are all 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 strain AH2012 / 12, PEDV N monoclonal antibody (PEDV N mAb), and African green monkey kidney cells (Vero) were provided by the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences. The BAC plasmid vector was stored in our laboratory. The pBAC-AH2012 / 12 infectious clone plasmid, which is the full-genome BAC recombinant plasmid of the AH2012 / 12 strain, was provided by the Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences and has been published in the literature "Yin Liping, Chang Changlin, Fan Baochao, et al. Construction of chimeric strains of porcine epidemic diarrhea virus variant strains and S-indel epidemic strains S gene [J / OL]. Chinese Veterinary Science, 1-8 [2025-04-30].". NEB 10-beta competent cells were purchased from Beijing Bomed Gene Technology Co., Ltd. 0.25% trypsin, fetal bovine serum (FBS), MEM (2×), and DMEM cell culture medium were purchased from Gibco; reverse transcriptase, 2× high-fidelity DNA polymerase (Phanta Max MasterMix), and total RNA mini extraction kit were purchased from Vazyme; 2× seamless cloning recombinase mixture (SeamLess CloningMix) was purchased from Beijing Bomed Gene Technology Co., Ltd.; DNA purification kit and gel extraction kit were provided by Guangzhou Omega; Large Construct Kit was purchased from QIAGEN; Cas 9 enzyme (Cas 9 nuclease. S.Pyogenes) and T7 RNA polymerase were purchased from NEB (Beijing) Co., Ltd. Transfection reagent Lipofectamine™ 3000, yeast extract, and tryptone were purchased from Thermo Fisher Scientific; DAPI staining solution and horseradish peroxidase-labeled goat anti-mouse IgG antibody were purchased from Beyotime; methylcellulose was purchased from Sigma. 15 six-week-old male BALB / c mice were purchased from the Animal Experiment Center of Yangzhou University. HRP-labeled goat anti-mouse IgG was purchased from abcam; single-component TMB chromogenic solution was purchased from Huzhou Yingchuang Biotechnology Co., Ltd.; sulfuric acid was purchased from Kelong Chemical Reagent Factory; Tween 20 was purchased from Solarbio.

[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. The double-stranded DNA forms of sgRNA-F and sgRNA-R were obtained by amplifying them with a scaffold oligonucleotide (scafold-oligo) respectively. The sgRNA-F and sgRNA-R were designed to perform PCR amplification on the scaffold oligonucleotide (scafold-oligo) to obtain the double-stranded DNA forms of sgRNA-F and sgRNA-R respectively. Among them, the scafold-oligo was used to provide the scaffold sequence part of 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 min, 95°C for 15 s, 60°C for 15 s, 72°C for 1 min, 35 cycles, 72°C for 5 min, and cycling at 4°C. Double-stranded DNA with a length of approximately 100 bp was generated. The amplification products were purified respectively using a DNA purification kit (Omegea Bio-Tek, Guangzhou, China) according to the 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, and specific primers C1347+1348S-M-F and C1347+1348S-M-R were designed to amplify fragment 2. Finally, using fragment 1 and fragment 2 as templates, the final target fragment was amplified with C1347+1348S-F and C1347+1348S-M-R. Finally, a pair of identification primers were designed to verify whether the strain was successfully mutated. The primer sequences are shown in Table 1.

[0052] Table 1 Primer sequences

[0053]

[0054] 1.3 Amplification of the target fragment containing the mutation

[0055] Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, fragment 1 was amplified with specific primers C1347+1348S-F and C1347+1348S-R, and fragment 2 was amplified with specific primers C1347+1348S-M-F and C1347+1348S-M-R. Finally, using fragment 1 and fragment 2 as templates, the final target fragment was amplified with specific primers C1347+1348S-F and C1347+1348S-M-R. The PCR program was: pre-denaturation at 95°C for 3 min, 95°C for 15 s, 60°C for 15 s, 72°C for 1 min, 35 cycles, 72°C for 5 min, and cycling at 4°C. The PCR system was: 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 In vitro cleavage of pBAC-AH2012 / 12 plasmid by CRISPR / Cas9

[0057] After in vitro transcription and purification of the double-stranded DNA product of the sgRNA obtained in part 1.2, the RNA product of the sgRNA was obtained.

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

[0059] 1.5 Homologous recombination and ligation transformation

[0060] Use homologous recombinase to ligate the DNA fragment containing a single-point mutation (i.e., the final target fragment amplified in part 1.3) and the linearized BAC plasmid after digestion and purification (i.e., the pBAC-AH2012 / 12 plasmid cut in part 1.4). The reaction system is as follows: 5 μL of 2× seamless cloning recombinase mixture, 2 μL of DNA fragment, and 3 μL of digestion product. The reaction condition is incubation at 50 °C for 20 min (a similar effect can be achieved with a time control of 15 - 30 min). After ligation, store it at -20 °C in the refrigerator for later use. Place the NEB10-beta competent cells in an ice-water mixture and let them thaw slowly. Under the environment of a laminar flow hood, add all the products of the ligation operation to the competent cells. Subsequently, gently tap the centrifuge tube to mix them evenly, and then place it in ice water and let it stand for 30 min. After standing, quickly transfer the competent cells to a water bath set at 42 °C for heat shock treatment for 50 s. Then place it back in the ice-water mixture for an ice bath for 2 min, add 600 μL of antibiotic-free liquid LB medium, and shake it on a shaker at 37 °C and 200 rpm for 1 h. Then centrifuge at 5000 rpm for 1 min, discard 500 μL of the LB medium, resuspend the bacterial pellet with the remaining liquid, and spread the bacterial solution on a solid agar plate. Let it stand. After the liquid is completely absorbed by the plate, invert it and place it in an incubator at 37 °C for overnight culture.

[0061] 1.6 Identification of bacterial liquid by PCR and extraction of recombinant plasmid

[0062] Pick single colonies from the plate, put them into LB with the corresponding antibiotic, shake the bacteria on a shaker at 37 °C and 200 rpm. After turbidity, perform bacterial liquid PCR using primers PEDV-F and PEDV-R. Send the PCR products with bands for sequencing by Sangon Biotech. Select the single colonies with correct sequencing identification for amplification and perform large-scale plasmid extraction according to the QIAGEN Large Construct kit instruction manual, measure the concentration, and store it at -80 °C.

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

[0064] One day in advance, seed Vero cells in a six-well plate. After growing to 70% confluence, transfect 6 μg of the recombinant plasmid extracted in part 1.6. After transfection, place the six-well plate in an incubator at 37 °C and 5% CO 2 and continue to culture. When cytopathic effect appears in the cells, freeze-thaw three times, centrifuge at 12000 rpm for 10 min, and collect the supernatant to obtain the recombinant virus, namely the porcine epidemic diarrhea virus with low replication ability (named r12-C1347+1348S), and store it at -80 °C.

[0065] 1.8 Indirect immunofluorescence assay

[0066] The 24-well plate filled with Vero cells was infected with the recombinant virus at an MOI of 0.01. After 24 h, the supernatant was discarded. The cells were fixed with a fixing solution (methanol:acetone = 1:4) for 15 min and then the solution was discarded; washed with PBS, 500 μL of mouse anti-PEDV N mAb (diluted 1:500) was added to each well, and after incubation at 37 °C for 1.5 h, the solution was discarded; washed with PBS, 500 μL of FITC-labeled goat anti-mouse IgG (diluted 1:2000) was added to each well, and incubated at 37 °C in the dark for 1 h; after washing 3 times with PBS, observed under a fluorescence microscope to determine the results.

[0067] 1.9 Plaque assay

[0068] Discard the culture medium of the Vero cells grown to confluence in the six-well plate, and wash the Vero cells twice with serum-free DMEM. Dilute the recombinant virus 10-fold respectively, from 10 -1 until 10 -5 . Take 500 μL of the virus solution at different dilution degrees to infect Vero cells in different wells. After incubation in a 37 °C incubator for 1.5 h, discard the virus solution, wash the Vero cells twice with DMEM, and finally add 2 mL of plaque fixing solution to cover the Vero cells. Culture in a 37 °C incubator for 24 - 48 h, and observe whether plaques appear on the cells every day. After the plaques appear, discard the plaque solution, wash twice with DMEM, and then add 1.5 mL of 0.1% crystal violet. Stain in the dark at 37 °C for 1 - 2 h, discard the crystal violet dye, and observe the plaque morphology.

[0069] 1.10 Determination of the 50% tissue culture infective dose (TCID 50 )

[0070] Take 100 μL of the virus solution, add 900 μL of DMEM maintenance solution, and dilute 10-fold serially to 10 -7 . Inoculate 100 μL into the Vero cells in a 96-well plate with good growth state, and set 8 replicates. After incubation for 1.5 h, discard the solution, supplement with 500 μL of DMEM maintenance solution, and observe the cytopathic effect within 72 h. Calculate the TCID 50 according to the Reed-Muench method.

[0071] 1.11 Growth curve identification

[0072] Vero cells in a 24-well plate were infected with the parental virus with an MOI of 0.01 and 10 recombinant viruses. The virus was harvested at four time points: 6 h, 12 h, 24 h, and 36 h after virus inoculation, and three replicate wells were set up. After incubation in a 37 °C incubator for 1.5 h, the supernatant of the virus solution was discarded, and the Vero cells were washed twice with DMEN. 500 μL of virus maintenance medium was added to each well of the 24-well plate, and the plate was placed back in the 37 °C incubator for further cultivation. The cell supernatants were collected at 6 h, 12 h, 24 h, and 36 h after infection, and their TCID 50 was measured. Based on the measured virus TCID 50 the growth curves of each virus strain were plotted.

[0073] 1.12 Design of the mouse experiment with recombinant viruses

[0074] The recombinant virus strain r12-C1347+1348S and the parental strain AH2012 / 12 were respectively inoculated into 6-week-old BALB / c mice. At the same time, a negative control group inoculated with DMEM was established to explore the changes in the immunogenicity of the virus with mutated palmitoylation sites of the PEDV S protein, so as to provide important clues for understanding the pathogenic mechanism of PEDV and developing new vaccines. The mouse experiment design is shown in Table 2. The parental strain AH2012 / 12 and the recombinant virus r12-C1347+1348S were both diluted to 1×10 5.50 TCID 50 / mL. After mixing with the adjuvant ISA206 on the immunization day, 300 μL was intramuscularly injected into each mouse. The MOCK group was intramuscularly injected with 300 μL of DMEM. Blood was collected before the first immunization to detect the antibody level in the mouse serum. The first immunization was recorded as day 0. Blood was collected on the 14th day after the first immunization for the second immunization, on the 28th day after the first immunization for the third immunization, on the 42nd day after the first immunization for the fourth immunization, and terminal blood collection and euthanasia were performed on the 56th day after the first immunization. The antibody level in the mouse serum was measured by ELISA.

[0075] Table 2 Mouse experiment design

[0076]

[0077] 2. Results

[0078] 2.1 Amplification of the target fragment containing the mutation

[0079] Using the pBAC-AH2012 / 12 infectious clone plasmid as a template, fragment 1 was amplified with specific primers C1347+1348S-F and C1347+1348S-R, and fragment 2 was amplified with specific primers C1347+1348S-M-F and C1347+1348S-M-R. Finally, using fragment 1 and fragment 2 as templates, the final target fragment was amplified with specific primers C1347+1348S-F and C1347+1348S-M-R. The length of the final target fragment is about 1000 bp ( Figure 1 ).

[0080] 2.2 In vitro cleavage of pBAC-AH2012 / 12 plasmid by CRISPR / Cas9

[0081] The pBAC-AH2012 / 12 infectious clone plasmid was cleaved in vitro using Cas9 nuclease from NEB. The results of agarose gel electrophoresis detection ( Figure 2 ) showed that a band of about 1000 bp was cleaved out.

[0082] 2.3 Identification of the bacterial liquid by PCR after transformation of homologous recombination products

[0083] After the amplified products containing mutations were cloned into the cleaved pBAC-AH2012 / 12 linear plasmid by homologous recombination, they were transformed into NEB 10-beta competent cells. After culturing, they were evenly spread on an LB solid plate containing 25 μg / mL chloramphenicol for screening. Five single colonies were picked and identified by PCR using PEDV-F and PEDV-R primers. The electrophoresis results ( Figure 3 ) showed that five colonies showed brighter bands around 1000 bp, which was consistent with the expectation.

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

[0085] The recombinant plasmid was transfected into Vero cells. After 36 h, syncytial lesions appeared in the cells ( Figure 4 ), indicating successful rescue. After extracting RNA and reverse transcribing it into cDNA, PCR amplification was carried out using the identification primers PEDV-F and PEDV-R. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The results ( Figure 5 ) showed that the sequence was correct, and cysteine at positions 1347 and 1348 was successfully mutated to serine. The results of indirect immunofluorescence assay confirmed that the virus was PEDV ( Figure 6 ).

[0086] 2.5 Comparison of biological characteristics of recombinant viruses

[0087] The results of plaque assay ( Figure 7showed that the ability of the recombinant virus r12-C1347+1348S to form plaques was relatively weaker than that of the parental virus AH2012 / 12, and the plaque area was smaller. The results of the growth curve detection ( Figure 8 showed that the virus titer of the recombinant virus r12-C1347+1348S decreased significantly compared with that of the parental virus AH2012 / 12, and decreased by about 2 titers at 12 hpi.

[0088] 2.6 Detection of mouse serum antibody levels

[0089] Blood was collected from the tail vein on the 14th, 28th, 42nd, and 56th days after immunization to prepare serum samples. Porcine epidemic diarrhea virus spike protein (PEDV S) was used to coat the antigen in a 96-well plate, and the dynamic changes of antibodies against S protein-specific immunoglobulin G (IgG) in the serum of experimental mice were quantitatively detected by indirect enzyme-linked immunosorbent assay (ELISA). The results ( 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 antibody levels induced by the recombinant virus r12-C1347+1348S began to exceed those of the parental virus rAH2012 / 12 42 days after immunization.

[0090] In summary, based on the infectious clone platform of the AH2012 / 12 strain, the present invention successfully constructed a recombinant virus r12-C1347+1348S with double-site mutations by using CRISPR / Cas9-mediated genome editing technology, and evaluated the immunogenicity of the recombinant virus through mouse experiments. The results showed that the recombinant virus r12-C1347+1348S could induce high levels of PEDV S protein IgG antibodies and neutralizing antibodies in mice, and the antibody levels produced exceeded those of its parental strain AH2012 / 12 28 days after immunization. These results indicate that the two palmitoylation modification sites mutated in r12-C1347+1348S can increase the antibody levels in mouse serum and are important targets for weakening the virus replication ability.

[0091] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined 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 1347th and 1348th amino acids 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 the digestion product; 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 reaction system of CRISPR / Cas9 in vitro cleavage 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 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 of 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 ability 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 described in claim 7.

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

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