PEDV s protein n-glycosylation site mutant virus and application thereof

Abstract

The application discloses a PEDV S protein N-glycosylation site mutant virus and application thereof. The application immunizes mice with the mutant virus to detect the influence of N-glycosylation of S protein on virus immunogenicity, and it is found that the recombinant PEDV virus strain with single-point mutation of N216, N324 and N1261 glycosylation sites of S protein can activate mice to produce a higher level of neutralizing antibody, and can be increased by 3.2 times (N216A), 6.7 times (N324A) and 3.2 times (N1261A). The recombinant PEDV virus strains with mutation of the three sites can be used as potential PEDV inactivated vaccine candidate strains.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a PEDV S protein N-glycosylation site mutant virus and application thereof. BACKGROUND

[0002] Coronaviruses belong to the order Nidovirales and the family Coronaviridae. The Coronaviridae family can be further divided into the subfamily Orthocoronavirinae and the subfamily Letovirinae, and the Orthocoronavirinae can be divided into the genera alphacoronavirus, betacoronavirus, deltacoronavirus and gammacoronavirus. Coronaviruses have a wide range of natural hosts, including mammals, birds and amphibians, and can cause respiratory, intestinal, liver and nervous system diseases, causing epidemics of epidemic diseases and threatening human and animal health.

[0003] Porcine epidemic diarrhea (PED) is a severe intestinal disease caused by porcine epidemic diarrhea virus (PEDV), and the main symptoms are diarrhea, vomiting and dehydration. Newborn piglets are susceptible to PEDV, and the mortality rate can reach 100%. In the 1970s, PEDV was first discovered in Europe. Since 2010, after the PEDV strain has been mutated, the highly pathogenic PEDV mutant strain has begun to spread widely in pig populations, causing huge economic losses to the global breeding industry. So far, PEDV is still the main pathogen causing viral diarrhea disease in pig populations, and its harm cannot be underestimated. Vaccination is one of the most important methods for preventing and controlling PED, and most of the conventional inactivated vaccines available at present have the advantages of high safety and simple production process. However, inactivated vaccines also have many disadvantages. During the preparation process, the immunogenicity may be reduced, and at the same time, since the inactivated vaccine cannot replicate in the host body, it needs to be added with adjuvants and high immune doses and multiple immunizations to improve the immune effect. In order to better prevent and control PED and protect the healthy breeding of pigs, how to improve the immunogenicity of the PEDV inactivated vaccine is of great significance.

[0004] Glycosylation is a post-translational modification of proteins, which refers to the process of attaching glycans to the polypeptide of a protein. So far, a variety of glycosylation modifications have been reported, including N-, O- and C-glycosylation modifications, glycosyl phosphatidylinositolization and phosphoglycosylation. Among them, N-glycosylation is the most common and widely studied form of protein glycosylation, and the conservative amino acid motif is N-X-S / T (X is all amino acids except proline), and the glycan will be attached to the amide nitrogen of the aspartic acid (N) residue. N-glycan of viral glycoprotein has various functions, including promoting protein expression, transportation, fusion, binding to cell surface receptors and preventing neutralization by antibodies, etc. SARS-CoV 2 S protein has high N-glycosylation modification, containing 22 N-glycosylation modification sites, and these glycosylation sites play an important role in its pathogenicity and immunogenicity. Many studies have shown that PEDV S protein is a highly glycosylated protein, which plays a crucial role in the immunogenicity and pathogenicity of PEDV. Therefore, it is still urgent to study the effect of N-glycosylation of S protein on the life cycle and immunogenicity of PEDV, so as to obtain more potential candidate strains of PEDV inactivated vaccine. SUMMARY

[0005] The technical problem to be solved by the present application is to provide an S protein with N-glycosylation site mutation.

[0006] The technical problem to be solved by the present application is to provide an S protein with N-glycosylation site mutation.

[0007] The technical problem to be solved by the present application is to provide an S protein with N-glycosylation site mutation.

[0008] The technical problem to be solved by the present application is to provide an S protein with N-glycosylation site mutation.

[0009] The technical problem to be solved by the present application is to provide an S protein with N-glycosylation site mutation.

[0010] The technical problem to be solved by the present application is to provide an S protein with N-glycosylation site mutation.

[0011] The technical problem to be solved by the present application is to provide an S protein with N-glycosylation site mutation.

[0012] The technical problem to be solved at last by the present application is to provide a PEDV neutralizing antibody.

[0013] Technical solution: In order to solve the above technical problems, the present application provides an N-glycosylation site mutant S protein, which is mutated on the PEDV S protein, and the amino acid sequence of the PEDV S protein is mutated from aspartic acid to alanine at positions 62, 118, 216, 264, 300, 324, 344, 351, 425, 514, 556, 667, 688, 726, 743, 781, 787, 873, 1009, 1196, 1232, 1249, 1261, 1273, 1278, 1295 or 1308 of the amino acid sequence of the PEDV S protein, and the nucleotide sequence of the PEDV S gene full length is shown in SEQ ID NO. 1.

[0014] A nucleic acid molecule encoding the N-glycosylation site mutant S protein, which is mutated on the PEDV S gene full length, and the nucleotide sequence of the PEDV S gene full length is shown in SEQ ID NO. 1.

[0015] Among them, the specific mutations of the PEDV S gene include: the 186-187th nucleotide, the 354-355th nucleotide, the 648-649th nucleotide, the 792-793th nucleotide, the 900-901th nucleotide, the 1032-1033th nucleotide, the 1053-1054th nucleotide, the 2001-2002th nucleotide, the 2064-2065th nucleotide, the 2229-2230th nucleotide, the 2343-2344th nucleotide, the 2619-2620th nucleotide, the 3027-3028th nucleotide, the 3588-3589th nucleotide, the 3696-3697th nucleotide, the 3783-3784th nucleotide, the 3819-3820th nucleotide, the 3834-3835th nucleotide or the 3885-3886th nucleotide of the PEDV S gene are mutated from AA to GC, the 972-974th nucleotide of the PEDV S gene is mutated from AAT to GCC, the 1275-1277th nucleotide of the PEDV S gene is mutated from AAT to GCG, the 1542-1543th nucleotide, the 1668-1669th nucleotide, the 2178-2179th nucleotide, the 2361-2362th nucleotide, the 3747-3748th nucleotide or the 3924-3925th nucleotide of the PEDV S gene are mutated from AA to GC.

[0016] The present application also includes an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacteria containing the nucleic acid molecule.

[0017] The present application also includes a recombinant PEDV infectious clone plasmid with S protein N-glycosylation site mutation, which is obtained by introducing the nucleic acid molecule into a vector.

[0018] The present application also includes a method for constructing the recombinant PEDV infectious clone plasmid with S protein N-glycosylation site mutation, which comprises the following steps:

[0019] (1) DNA fragment amplification: amplifying a DNA fragment of PEDV S gene containing the following mutation sites, wherein the DNA fragment is mutated from AA to GC at the 186-187th, 354-355th, 648-649th, 792-793th, 900-901th, 1032-1033th, 1053-1054th, 2001-2002th, 2064-2065th, 2229-2230th, 2343-2344th, 2619-2620th, 3027-3028th, 3588-3589th, 3696-3697th, 3783-3784th, 3819-3820th, 3834-3835th or 3885-3886th nucleotide of the PEDV S gene, the DNA fragment is mutated from AAT to GCC at the 972-974th nucleotide of the PEDV S gene, the DNA fragment is mutated from AAT to GCG at the 1275-1277th nucleotide of the PEDV S gene, the DNA fragment is mutated from AA to GC at the 1542-1543th nucleotide of the PEDV S gene, the DNA fragment is mutated from AA to GC at the 1668-1669th nucleotide of the S gene, the DNA fragment is mutated from AA to GC at the 2178-2179th nucleotide of the S gene, the DNA fragment is mutated from AA to GC at the 2361-2362th nucleotide of the S gene, the DNA fragment is mutated from AA to GC at the 3747-3748th nucleotide or 3924-3925th nucleotide of the S gene;

[0020] (2) amplification of the targeting fragment: amplifying the first part of the targeting fragment and the second part of the targeting fragment respectively using the DNA fragment of step (1) as a template, then amplifying the middle part, and finally connecting the three fragments to obtain the targeting fragment;

[0021] (3) first Red recombination: transforming the targeting fragment into competent cells and verifying to obtain positive clones;

[0022] (4) The second step of Red recombination: picking positive clones for temperature and L-arabinose induction, and finally obtaining the final positive clones through antibiotic screening.

[0023] The present application also includes a PEDV S protein N-glycosylation site mutant virus, which is obtained by transfecting cells with the recombinant PEDV infectious clone plasmid.

[0024] The present application also includes a rescue method for the PEDV S protein N-glycosylation site mutant virus, which comprises the following steps: co-transfecting cells with the infectious clone plasmid and a helper plasmid.

[0025] The present application also includes the use of the N-glycosylation site mutant S protein, the nucleic acid molecule, the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacteria, the S protein N-glycosylation site mutant recombinant PEDV infectious clone plasmid or the PEDV S protein N-glycosylation site mutant virus in the preparation of a PEDV vaccine or a PEDV antibody.

[0026] The present application also includes a PEDV vaccine containing the N-glycosylation site mutant S protein, the nucleic acid molecule, the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacteria, the S protein N-glycosylation site mutant recombinant PEDV infectious clone plasmid or the PEDV S protein N-glycosylation site mutant virus.

[0027] The present application also includes a PEDV neutralizing antibody, which is obtained by infecting animals with the PEDV S protein N-glycosylation site mutant virus.

[0028] In order to study the effect of N-glycosylation of S protein on the life cycle and immunogenicity of PEDV, the present application first predicts the N-glycosylation modification sites of PEDV GX4 / 2021 S protein using the online tool NetNGlyc-1.0. GX4 / 2021 S protein has a total of 27 potential N-glycosylation modification sites. The 27 N-glycosylation single-point mutant PEDV infectious clone plasmids are constructed using Red recombination technology, and the virus is rescued. The results show that the 873 N-glycosylation site is crucial for virus rescue. Finally, the effect of S protein N-glycosylation on viral immunogenicity is explored, and the most comprehensive PEDV S protein N-glycosylation site single-point mutant virus library is successfully obtained.

[0029] Beneficial effects: The present application detects the influence of N-glycosylation on the immunogenicity of the mutant virus by immunizing mice with the mutant virus, and the results show that the recombinant PEDV virus strains with glycosylation site mutations at positions N216, N324 and N1261 of the S protein can activate mice to produce higher levels of neutralizing antibodies, which can be increased by 3.2 times (N216A), 6.7 times (N324A) and 3.2 times (N1261A). The three mutant recombinant PEDV virus strains can be used as potential PEDV inactivated vaccine candidate strains. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the prediction result of the N-glycosylation site of PEDV GX4 / 2021 S protein;

[0031] Figure 2 is the homology modeling of PEDV GX4 / 2021 S protein;

[0032] Figure 3 is the Sanger sequencing result of the PEDV S protein N-glycosylation site mutant infectious clone plasmid;

[0033] Figure 4 is the cytopathic effect of PEDV recombinant virus;

[0034] Figure 5 is the IFA identification of PEDV recombinant virus;

[0035] Figure 6 is the western blot identification of PEDV recombinant virus;

[0036] Figure 7 is the TCID50 determination of PEDV recombinant virus;

[0037] Figure 8 is the identification of adsorption, invasion and release of recombinant PEDV;

[0038] Figure 9 is the determination of neutralizing antibodies in mouse serum. DETAILED DESCRIPTION

[0039] The present application will be further described below in combination with the drawings and specific embodiments.

[0040] 1. Experimental materials

[0041] PEDV strain PEDV GX4 / 2021 (GenBank accession number: OP382083) was isolated, purified and preserved by the laboratory. Vero cells, pET30a, pBAC-PEDV, E. coli GS1783-pBAC-PEDV GX4 / 2021 strain, pCAGGS-PEDV N were preserved by the laboratory. (The above plasmids and strains were published in Chinese Patent Publication No. CN116445528A Construction Method of Recombinant Porcine Epidemic Diarrhea Virus Infectious Clone, Infectious Clone and Application Thereof) Anti-PEDV-N monoclonal antibody, anti-PEDV-S monoclonal antibody were prepared and preserved by the laboratory. Goat anti-mouse IgG (H+L)-HRP, anti-GAPDH monoclonal antibody were purchased from Wuhan ABclonal Company; ECL luminous liquid was purchased from Suzhou Xin Saimei Biological Technology Co., Ltd. 6-8 week-old BALB / c female mice were purchased from the Experimental Animal Center of Comparative Medical College of Yangzhou University.

[0042] 2. Analysis of N-glycosylation modification sites of PEDV GX4 / 2021 S protein

[0043] The N-glycosylation modification sites of PEDV GX4 / 2021 S protein were predicted using online tool NetNGlyc-1.0 (https: / / services.healthtech.dtu.dk / services / NetNGlyc-1.0 / ). The results showed that GX4 / 2021 strain S protein had a total of 27 potential N-glycosylation modification sites, S1 region contained 15 N-glycosylation sites, S2 region contained 12 N-glycosylation sites. Figure 1 ) N-glycosylation sites were distributed in S1 region as follows: D0 domain contained 3 N-glycosylation sites, NTD domain contained 6 N-glycosylation sites, SD1 domain did not contain predicted N-glycosylation sites, CTD domain contained 2 N-glycosylation sites, and SD2 domain contained 4 N-glycosylation sites.

[0044] 3. Homology modeling of PEDV GX4 / 2021 S protein

[0045] Homology modeling was performed on PEDV GX4 / 2021 S protein trimer using online tool SWISS-MODEl (https: / / swissmodel.expasy.org / ), in which the protein model used was PEDV Pintung 52 S protein with PDB number: 7w6m. Finally, the annotation of N-glycosylation sites was performed. The results showed that 22 predicted N-glycosylation sites were located on the surface of the S protein trimer. Among them, the N-glycosylation sites located in the S1 region were all exposed on the surface of the S protein trimer. Due to the function of the S2 subunit and the correct folding of the S protein, the N-glycosylation sites near the HR2-TM region were inside the S protein, and the remaining N-glycosylation sites were all located on the surface of the S protein. Figure 2

[0046] 4. Construction of recombinant virus plasmid of PEDV S protein N-glycosylation site mutation

[0047] According to the prediction results of S protein N-glycosylation sites, single-point mutant PEDV GX4 / 2021 recombinant virus plasmid was constructed, and the mutation rule of N-glycosylation site was that the asparagine (Asn, N) in the Asn-X-Ser / Thr (X is any amino acid except proline) motif was mutated to alanine (Ala, A). The primer sequences are shown in Table 1.

[0048] Table 1 PEDV S gene N-glycosylation site mutation primer

[0049]

[0050]

[0051]

[0052] Red recombination technology was used to mutate individual N-glycosylation sites, and PE-nsp16 F1, PE-S-cR2, I-SceI-CAT F1 and CATpro-Kan R were universal primers. The construction process of the recombinant virus infectious clone plasmid pBAC-PEDV GX4 / 2021-S-N62A, in which the 62th aspartic acid of the S gene was mutated to alanine, was taken as an example, and the specific steps were as follows:

[0053] ​(1) Amplification of DNA fragment S-N62A. The fragment S-N62A-F was amplified using the primer pair PE-nsp16 F1 and PE-S N62AR1 with the reaction system of Table 2, and the fragment S-N62A-R was amplified using the primer pair PE-SN62A F1 and PE-S-c R2 with the reaction system of Table 2, with pBAC-PEDV as template 1. The fragment S-N62A was amplified using the primer pair PE-nsp16 F1 and PE-S-c R2 with the reaction system of Table 3 by overlap PCR, with the fragment S-N62A-F and the fragment S-N62A-R as template 2 and template 3. All PCR reactions were performed according to the conditions of Table 5.

[0054] Table 2 PCR reaction system

[0055]

[0056]

[0057] Table 3 PCR reaction system

[0058] ddH2O q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix

[0059] Table 4

[0060] ddH2O 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL

[0061] Table 5 PCR amplification conditions

[0062]

[0063] (2) Amplification of the targeting fragment b-S-N62A-Kan-c. The first part of the targeting fragment b-S-N62A was amplified using the primer pair PE-nsp16 F1 and PE-S N62AR2 with the reaction system of Table 2, and the second part of the targeting fragment S-N62A-c was amplified using the primer pair PE-S N62A F2 and PE-S-c R2 with the reaction system of Table 2, with the fragment S-N62A as template 4. The middle part of the targeting fragment Kan was amplified using the primer pair I-SceI-CAT F1 and CATpro-Kan R with pET30a as template. The targeting fragment b-S-N62A-Kan-c was amplified using the primer pair PE-nsp16 F1 and PE-S-c R2 with the reaction system of Table 4 by three-fragment overlap PCR, with the fragment b-S-N62A, the fragment Kan and the fragment S-N62A-c as template 5, template 6 and template 7. All PCR reactions were performed according to the conditions of Table 5.

[0064] (3) First step Red recombination. The targeting fragment b-S-N62A-Kan-c was transformed into GS1783-pBAC-PEDV GX4 / 2021 competent cells by electroporation, and the competent cells were coated on LB plates containing chloramphenicol and kanamycin double resistance and cultured at 32°C for 24h to perform the first step Red recombination.

[0065] (4) Single colonies were picked from the plates, and colony PCR identification was performed using primer pair PE-nsp16 F1 and PE-S-c R2. The correct positive colonies were streaked on plates containing kanamycin resistance and cultured at 32°C for 16h. Colony PCR identification was again performed using primer pair PE-nsp16 F1 and PE-S-c R2, and the purified positive colonies were named pBAC-PEDV GX4 / 2021-S-N62A-Kan.

[0066] (5) Second step Red recombination. A positive single colony was inoculated into 2mL LB liquid medium containing chloramphenicol and cultured at 32°C on a shaker until turbidity. 2mL LB liquid medium containing 2% L-arabinose and chloramphenicol was added, and the culture was continued for 1h. Then it was transferred to a shaker at 42°C for 30min, and then to a shaker at 32°C for 3h.

[0067] (6) 100μL of the bacterial solution was taken for 10 4 fold dilution, and 200μL of the diluted bacterial solution was spread on LB plates containing 1% L-arabinose and chloramphenicol. After the bacterial solution was fully absorbed, the plates were inverted and incubated at 32°C for about 24h.

[0068] (7) Single colonies obtained in step (6) were spotted on LB plates containing chloramphenicol and kanamycin. Colonies that did not grow on kanamycin LB solid medium and grew on chloramphenicol LB solid medium were successfully recombined, and the obtained positive clone was named pBAC-PEDV GX4 / 2021-S-N62A. DNA sequencing was performed to verify whether the recombinant plasmid was successfully constructed.

[0069] The mutation process at other sites is basically the same as that at the 62nd amino acid site of the S protein, except for the replacement of the mutation primer:

[0070] For example: construction of pBAC-PEDV GX4 / 2021-S-N216A: the primers used are PE-S N216A F1, PE-S N216A R1, PE-S N216A R2 and PE-S N216A F2. The specific steps are: using pBAC-PEDV as template 1, using the reaction system in Table 2, using primer pair PE-nsp16 F1 and PE-S N216A R1 to amplify fragment S-N216A-F, using the reaction system in Table 2, using primer pair PE-S N216A F1 and PE-S-c R2 to amplify fragment S-N216A-R; using fragment S-N216A-F and fragment S-N216A-R as template 2 and template 3, using the reaction system in Table 3, using primer pair PE-nsp16 F1 and PE-S-c R2 to amplify fragment S-N216A by overlap PCR. All PCR reactions are performed according to the conditions in Table 5. Amplification of targeting fragment b-S-N216A-Kan-c. Using fragment S-N216A as template 4, using the reaction system in Table 2, using primer pair PE-nsp16 F1 and PE-S N216A R2 to amplify the first part of the targeting fragment b-S-N216A, using primer pair PE-S N216A F2 and PE-S-c R2 to amplify the second part of the targeting fragment S-N216A-c, using pET30a as template, using primer pair I-SceI-CAT F1 and CATpro-Kan R to amplify the middle part of the targeting fragment Kan. Using fragment b-S-N216A, fragment Kan and S-N216A-c as template 5, template 6 and template 7, according to the PCR reaction system in Table 4, using primer pair PE-nsp16 F1 and PE-S-c R2, using three-fragment overlap PCR to amplify the targeting fragment b-S-N216A-Kan-c. All PCR reactions are performed according to the conditions in Table 5.

[0071] The construction methods of the remaining recombinant plasmids are the same as above.

[0072] Construction of pBAC-PEDV GX4 / 2021-S-N118A: the primers used are PE-S N118A F1, PE-S N118A R1, PE-S N118A R2 and PE-S N118A F2.

[0073] Construction of pBAC-PEDV GX4 / 2021-S-N264A: the primers used are PE-S N264A F1, PE-S N264A R1, PE-S N264A R2 and PE-S N264A F2.

[0074] Construction of pBAC-PEDV GX4 / 2021-S-N300A: Primers used were PE-S N300A F1, PE-S N300A R1, PE-S N300A R2, and PE-S N300A F2.

[0075] Construction of pBAC-PEDV GX4 / 2021-S-N324A: Primers used were PE-S N324A F1, PE-S N324A R1, PE-S N324A R2, and PE-S N324A F2.

[0076] Construction of pBAC-PEDV GX4 / 2021-S-N344A: Primers used were PE-S N344A F1, PE-S N344A R1, PE-S N344A R2, and PE-S N344A F2.

[0077] Construction of pBAC-PEDV GX4 / 2021-S-N351A: Primers used were PE-S N351A F1, PE-S N351A R1, PE-S N351A R2, and PE-S N351A F2.

[0078] Construction of pBAC-PEDV GX4 / 2021-S-N425A: Primers used were PE-S N425A F1, PE-S N425A R1, PE-S N425A R2, and PE-S N425A F2.

[0079] Construction of pBAC-PEDV GX4 / 2021-S-N514A: Primers used were PE-S N514A F1, PE-S N514A R1, PE-S N514A R2, and PE-S N514A F2.

[0080] Construction of pBAC-PEDV GX4 / 2021-S-N556A: Primers used were PE-S N556A F1, PE-S N556A R1, PE-S N556A R2, and PE-S N556A F2.

[0081] Construction of pBAC-PEDV GX4 / 2021-S-N667A: Primers used were PE-S N667A F1, PE-S N667A R1, PE-S N667A R2, and PE-S N667A F2.

[0082] Construction of pBAC-PEDV GX4 / 2021-S-N688A: Primers used were PE-S N688A F1, PE-S N688A R1, PE-S N688A R2, and PE-S N688A F2.

[0083] Construction of pBAC-PEDV GX4 / 2021-S-N726A: Primers used were PE-S N726A F1, PE-S N726A R1, PE-S N726A R2, and PE-S N726A F2.

[0084] Construction of pBAC-PEDV GX4 / 2021-S-N743A: Primers used were PE-S N743A F1, PE-S N743A R1, PE-S N743A R2, and PE-S N743A F2.

[0085] Construction of pBAC-PEDV GX4 / 2021-S-N781A: Primers used were PE-S N781A F1, PE-S N781A R1, PE-S N781A R2, and PE-S N781A F2.

[0086] Construction of pBAC-PEDV GX4 / 2021-S-N787A: Primers used were PE-S N787A F1, PE-S N787A R1, PE-S N787A R2, and PE-S N787A F2.

[0087] Construction of pBAC-PEDV GX4 / 2021-S-N873A: Primers used were PE-S N873A F1, PE-S N873A R1, PE-S N873A R2, and PE-S N873A F2.

[0088] Construction of pBAC-PEDV GX4 / 2021-S-N1009A: Primers used were PE-S N1009A F1, PE-S N1009A R1, PE-S N1009A R2, and PE-S N1009A F2.

[0089] Construction of pBAC-PEDV GX4 / 2021-S-N1196A: Primers used were PE-S N1196A F1, PE-S N1196A R1, PE-S N1196A R2, and PE-S N1196A F2.

[0090] Construction of pBAC-PEDV GX4 / 2021-S-N1232A: the primers used are PE-S N1232A F1, PE-S N1232A R1, PE-S N1232A R2 and PE-S N1232A F2.

[0091] Construction of pBAC-PEDV GX4 / 2021-S-N1249A: the primers used are PE-S N1249A F1, PE-S N1249A R1, PE-S N1249A R2 and PE-S N1249A F2.

[0092] Construction of pBAC-PEDV GX4 / 2021-S-N1261A: the primers used are PE-S N1261A F1, PE-S N1261A R1, PE-S N1261A R2 and PE-S N1261A F2.

[0093] Construction of pBAC-PEDV GX4 / 2021-S-N1273A: the primers used are PE-S N1273A F1, PE-S N1273A R1, PE-S N1273A R2 and PE-S N1273A F2.

[0094] Construction of pBAC-PEDV GX4 / 2021-S-N1278A: the primers used are PE-S N1278A F1, PE-S N1278A R1, PE-S N1278A R2 and PE-S N1278A F2.

[0095] Construction of pBAC-PEDV GX4 / 2021-S-N1295A: the primers used are PE-S N1295A F1, PE-S N1295A R1, PE-S N1295A R2 and PE-S N1295A F2.

[0096] Construction of pBAC-PEDV GX4 / 2021-S-N1308A: the primers used are PE-S N1308A F1, PE-S N1308A R1, PE-S N1308A R2 and PE-S N1308A F2.

[0097] The constructed recombinant plasmids were subjected to Sanger sequencing, and as shown in Table 1, all the 27 plasmids of S protein N-glycosylation sites constructed by the application were successfully constructed. q-PCR reaction mix

[0098] 5、Rescue of PEDV mutant virus

[0099] ​(1) The positive colonies of the 27 PEDV infectious clone plasmids containing single-point mutations of the S protein N-glycosylation site were expanded and cultured, and the plasmids were extracted using a plasmid extraction kit.

[0100] (2) The Vero cells in good growth state were uniformly plated in a 6-well plate, and when the cell density reached 70%-80%, the transfection was performed according to the instructions of the 3000 reagent.

[0101] (3) The amount of plasmid per well was as follows: 0.2 μg of auxiliary plasmid pCAGGS-PEDV-N and 1.8 μg of PEDV infectious clone plasmid with S protein N-glycosylation site mutation. The wild-type PEDV infectious clone plasmid (pBAC-PEDV) was used as a positive control. After 4-6 h of transfection, the cell culture medium was replaced with fresh DMEM medium containing 10% FBS, and after 24 h of transfection, the cell culture supernatant was discarded, the cell monolayer was washed twice with sterilized PBS buffer (0.01 M, pH = 7.2), and 2 mL of DMEM medium containing 2 μg / mL trypsin was added. After 2 days, the cell supernatant was collected, and fresh Vero cells were infected again.

[0102] (4) When the cells showed typical cytopathic lesions, it was determined that the recombinant virus rescue was successful, and it was marked as P0 generation. The rescued virus was continuously passaged in Vero cells, and each generation of virus liquid was marked and stored at -80°C.

[0103] As shown in 1 μL , the S protein N873 site mutant PEDV recombinant virus did not show typical cytopathic cell lesions in Vero cells, and the recombinant rescue failed. The remaining 26 N-glycosylation site single-point mutant viruses showed typical cytopathic lesions after infecting Vero cells, indicating that the 26 recombinant viruses were successfully rescued.

[0104] 6. Identification of recombinant virus

[0105] 6.1 Indirect immunofluorescence identification

[0106] The parent virus rGX4 / 2021 and the S protein N-glycosylation site mutant virus were infected into Vero cells, and the cells were fixed after 24 h. The monoclonal antibody against PEDV-N protein was used for detection. The specific steps were as follows:

[0107] ​(1) Vero cells in good growth condition were evenly spread into a 12-well plate. When the cell density reached 90%, the cell monolayer was washed twice with sterilized PBS buffer (0.01 M, pH = 7.2). 300 μL of the P0 generation rescue virus solution was inoculated into the cell monolayer. At the same time, a well without virus solution was set up. After incubation at 37°C, 5% CO2 constant temperature incubator for 2 h, the supernatant was discarded, and DMEM medium containing 2 μg / mL trypsin was added. The culture was continued for 24 h, and the cell culture supernatant was collected and marked as P1 generation.

[0108] (2) The cell monolayer was washed once with PBS, fixed with pre-cooled 4% paraformaldehyde at room temperature for 10 min, and washed twice with PBS.

[0109] (3) Add pre-cooled PBS solution containing 0.1% Triton X-100 and 2% BSA, permeabilize and block the membrane at room temperature for 30 minutes, and wash twice with PBS.

[0110] (4) Add anti-PEDV-N monoclonal antibody diluted 1:2000, incubate at 37°C for 1 h, and wash three times with PBS.

[0111] (5) Add 1:2000 diluted DyLight 488-labeled goat anti-mouse IgG secondary antibody, incubate at 37°C in the dark for 1 h, and wash three times with PBS.

[0112] (6) Add 1:10 diluted DAPI staining solution, incubate at room temperature for 5 min, wash three times with PBS, add appropriate amount of PBS, observe and photograph under an inverted fluorescence microscope.

[0113] like q-PCR reaction mix As shown, in addition to the rGX4 / 2021-S-N873A infected cell group, another 26 PEDV recombinant viruses with mutations in the N-glycosylation sites of the S protein had green fluorescence, indicating that 26 PEDV mutant viruses were successfully rescued.

[0114] 6.2 Western blot identification of recombinant viruses

[0115] (1) Vero cells were plated in 12-well plates in advance and cultured until the cell density reached 90%.

[0116] (2) Vero cells were infected with the parental virus rGX4 / 2021 and the 26 successfully rescued PEDV mutant viruses at an MOI of 0.01 for 24 h. A virus-uninfected group was set as a negative control.

[0117] (3) Discard the culture supernatant, wash twice with PBS buffer, add 150 μL of RIPA lysis buffer to each well, and collect the protein sample.

[0118] (4) The collected protein samples were subjected to western blot identification. The primary antibodies used were anti-PEDV N protein monoclonal antibody and anti-PEDV S protein monoclonal antibody, and the secondary antibody used was goat anti-mouse IgG (H+L)-HRP.

[0119] The results are as follows 1 μL As shown, the expression of N protein and S protein can be detected in both virus-infected groups, indicating that the mutation of the N-glycosylation site of S protein does not affect the expression of S protein.

[0120] 6.3 TCID of recombinant virus 50 Determination of

[0121] The successfully rescued virus was infected into Vero cells for 24 h, and the supernatant was collected and analyzed by TCID 50 Determination of q-PCR reaction mix As shown, after mutation of different N-glycosylation sites of S protein, the TCID 50 The N-glycosylation site mutation in the S1 subunit has a significant effect on the TCID of PEDV. 50 The greatest impact. TCID of the parent virus rGX / 2021 50 In comparison, after the N351 site mutation, the TCID 50 It can be reduced by 3000 times. After the N216 site mutation, the TCID of the recombinant virus 50 It can be reduced by 1000 times. After the mutation of N264, N324, and N688 sites, the TCID of the recombinant virus 50 The TCID50 of the recombinant virus decreased by 100 times after the mutation of N118 and N425. In the S2 subunit, the mutation of N787 had a negative effect on the TCID50 of the recombinant virus. 50 The impact was the greatest, decreasing by about 15 times. After mutations at N726, N1249, N1261, N1273, N1278, and N1308, the TCID 50 It dropped by about 5 to 10 times.

[0122] 7. Characterization of PEDV S protein N-glycosylation site mutant viruses

[0123] 7.1 Virus adsorption test

[0124] (1) Vero cells were evenly plated in a 24-well plate. When the cell density reached 90%, the cell culture medium was discarded and the cells were washed twice with PBS buffer.

[0125] (2) Recombinant PEDV viruses with mutations in the N-glycosylation sites of the S1 region (PEDV recombinant viruses with mutations in aspartic acid at positions 62, 118, 216, 264, 300, 324, 344, 425, 514, 556, 667, 688, or 743 of the S protein) were diluted with DMEM medium (company: Gibco, product number: 11965092) containing 2 μg / mL trypsin and added to the cell monolayer and placed at 4°C for 1 h.

[0126] (3) Remove the cells, discard the supernatant, wash the cells twice with pre-cooled PBS buffer, and collect the cell sample.

[0127] (4) Total cellular RNA was extracted using the RNA-easy extraction kit from Novozymes according to the instructions, and then reverse transcribed to obtain cDNA. According to the instructions of the qPCR kit from Yisheng Biotechnology (Cat. No. 11201ES08), relative fluorescence quantitative PCR was used to detect the effect of the removal of N-glycosylation of PEDV GX4 / 2021 on its adsorption efficiency. The PEDV-N gene was used to detect the content of PEDV in cells. GAPDH was a housekeeping gene. The quantitative PCR primers are shown in Table 6.

[0128] Table 6 Quantitative PCR primers

[0129] 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix

[0130] 7.2 Virus Invasion Test

[0131] (1) After the virus adsorption test is completed, the cell plate is removed, the supernatant is discarded, and the cells are washed twice with pre-cooled PBS buffer and coronavirus culture medium is added.

[0132] (2) Place the cell plate in a 37°C cell culture incubator and let it stand for 1 hour.

[0133] (3) Remove the cell plate, knock off the supernatant, wash the cells twice with pre-cooled PBS buffer, and collect the cell sample.

[0134] (4) Same as step (4) in 7.1.

[0135] 7.3 Viral Replication

[0136] The mutant virus with mutation of N-glycosylation site in S1 region was inoculated into Vero cells and ST cells at MOI=0.01, and the virus solution was collected after 24 hours for TCID 50 Determination of.

[0137] Previous experimental results show that PEDV S protein has N-glycosylation modification, and this N-glycosylation modification affects the rescue of the virus and the titer of the virus. In order to explore the impact of these N-glycosylation site mutations on the stage of the virus life cycle, we conducted relevant experiments on the effects of S1 subunit N-glycosylation site mutations on the adsorption, invasion and release stages of the virus. For the invasion stage of the virus, in Vero cells, mutations at N514, N556 in the CTD region and N688 in the SD2 region significantly inhibited the adsorption efficiency of the recombinant virus ( 1 μL A) In ST cells, mutations at N556, N688, N726, N743, and N216 inhibited the virus's adsorption efficiency to ST cells ( q-PCR reaction mix D) During the virus invasion phase, mutations at N514, N556, and N688 sites significantly inhibited the virus's invasion of Vero cells ( 1 μL B), mutations at N556, N688, N324, N300, and N216 inhibit viral invasion of ST cells ( q-PCR reaction mix E). After the virus invades the cell, it uses the cell's nutrients to complete its own proliferation. Whether in Vero cells or ST cells, mutations at N216, N324, N514, or N688 significantly inhibit the virus's replication in the cell ( 1 μL C and 8F).

[0138] 8. Animal testing

[0139] 8.1 Immunization of mice

[0140] In order to explore the effect of N-glycosylation modification of S protein on viral immunogenicity, the characteristics of the mutant virus and the highest TCID that can be amplified were comprehensively considered. 50 We selected five PEDV S protein N-glycosylation site mutant viruses (rGX4 / 2021-S-N118A, rGX4 / 2021-S-N216A, rGX4 / 2021-S-N324A, rGX4 / 2021-S-N1009A, and rGX4 / 2021-S-N1261A) and immunized mice 2 weeks apart according to the experimental protocol in Table 7. Mice immunized with rGX4 / 2021 served as the positive control group. Blood was collected 10 days after the fourth immunization for antibody titer.

[0141] Table 7 Experimental design of mice immunized with PEDV S protein N-glycosylation site mutant virus

[0142]

[0143] 8.2 Determination of neutralizing antibodies in mouse serum

[0144] (1) Vero cells were plated into 96-well plates one day in advance and the experiment was carried out when the cell density reached 90%.

[0145] (2) The serum after the fourth immunization was inactivated at 56°C for 30 min and filtered using a 0.22 μm filter in a clean bench.

[0146] (3) The serum was diluted 2-fold with DMEM medium. 250 μL of diluted antibody and 250 μL of diluted PEDV GX4 / 2021 virus solution (100 TCID 50 / 100 μL) and mix thoroughly, and incubate at 37°C for 1 h.

[0147] (4) After the incubation, discard the culture medium in the 96-well plate and add 100 μL of serum-virus mixture to the cell wells. Make 4 parallels for each dilution and set up a positive control well (add only 100 TCID 50 Virus solution) and negative control wells (only DMEM medium) were added, and the cells were incubated in a 5% CO2, 37°C incubator for 1 h.

[0148] (5) After the reaction is completed, the supernatant is discarded and the cells are washed twice with PBS. 100 μL of coronavirus culture medium containing 2 μg / mL trypsin is added to the cell wells and culture is continued for 48 h. The neutralizing titer of the immunized mouse serum is calculated. The calculation formula is: lg (neutralizing antibody titer) = L + d(S-0.5). (L: logarithm of the highest serum dilution; d: group distance, i.e., dilution coefficient, which is -0.3 for a 2-fold serial dilution; S: the sum of the ratios of the number of CPE wells to the number of inoculated wells in each group.)

[0149] We used virus neutralization assay to detect the level of neutralizing antibodies against PEDV in the serum of immunized mice. q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q-PCR reaction mix 1 μL q- As shown, mice in the DMEM immunization group could not produce neutralizing antibodies against PEDV, while mice immunized with rGX4 / 2021 produced neutralizing antibodies with a titer of 2 against PEDV. 3.4 , N216, N324, and N1261 mutations can significantly enhance the production of neutralizing antibodies against PEDV in PEDV-induced mice. The neutralizing antibody titers against PEDV produced by mice immunized with rGX4 / 2021-S-N216A, rGX4 / 2021-S-N324A, and rGX4 / 2021-S-N1261A were 2 5 , 2 6 , 2 5 , the neutralizing antibody titer increased by 3.2 times, 6.7 times, and 3.2 times, respectively.

[0150] SEQ ID NO.1:

[0151]

[0152]

[0153] SEQ ID NO. 2:

[0154]

[0155]

Claims

1. A S-protein mutated at an N-glycosylation site, characterized in that, The N-glycosylation site mutant S protein is mutated on the PEDV S protein, and the aspartic acid at position 324 of the amino acid sequence of the PEDV S protein is mutated to alanine, and the amino acid sequence of the PEDV S protein is shown as SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encoding the N-glycosylation site mutant S protein of claim 1.

3. Expression cassette, recombinant vector, recombinant cell or recombinant bacteria, characterized in that, The nucleic acid molecule of claim 2.

4. A recombinant PEDV infectious clone plasmid of S protein N-glycosylation site mutation, characterized in that, The recombinant PEDV infectious clone plasmid is obtained by introducing the nucleic acid molecule of claim 2 into a vector.

5. The method for constructing a recombinant PEDV infectious clone plasmid mutated at the N-glycosylation site of S protein according to claim 4, characterized in that, The method comprises the following steps: (1) DNA fragment amplification: amplifying the DNA fragment of the nucleic acid molecule of claim 2; (2) amplification of the targeting fragment: amplifying the first part of the targeting fragment and the second part of the targeting fragment using the DNA fragment of step (1) as a template, then amplifying the middle part, and finally connecting the three fragments to obtain the targeting fragment; (3) First step Red recombination: transform the targeting fragment into competent cells and verify to obtain positive clones; (4) Second step Red recombination: pick positive clones for temperature and L-arabinose induction, and finally further obtain the final positive clones through antibiotic screening.

6. A PEDV S protein N-glycosylation site mutant virus, characterized in that, The PEDV S protein N-glycosylation site mutant virus is obtained by transfecting cells with the recombinant PEDV infectious clone plasmid of claim 4 or 5.

7. The method for rescuing PEDV S protein N-glycosylation site mutant virus according to claim 6, characterized in that, The method comprises the following steps: co-transfecting cells with the infectious clone plasmid of claim 4 and the helper plasmid to obtain the PEDV S protein N-glycosylation site mutant virus.

8. Use of the N-glycosylation site mutant S protein of claim 1, the nucleic acid molecule of claim 2, the expression cassette of claim 3, the recombinant vector, the recombinant cell or the recombinant bacteria, the S protein N-glycosylation site mutant recombinant PEDV infectious clone plasmid of claim 4, or the PEDV S protein N-glycosylation site mutant virus of claim 6 in the preparation of a PEDV vaccine.

9. A PEDV vaccine, characterized in that, The nucleic acid molecule of claim 2, the expression cassette of claim 3, the recombinant vector, the recombinant cell or the recombinant bacteria, the S protein N-glycosylation site mutant recombinant PEDV infectious clone plasmid of claim 4, or the PEDV S protein N-glycosylation site mutant virus of claim 6.

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