Chimeric PEDV infectious clone plasmid, PEDV chimeric virus and construction method and application thereof

Abstract

The invention discloses a chimeric PEDV (porcine epidemic diarrhea virus) infectious clone plasmid, a PEDV chimeric virus as well as a construction method and application thereof. According to the invention, a PEDV genome is used as a skeleton for the first time, the chimeric virus for expressing the extracellular domain of the TGEV S protein is successfully rescued, and it is found that the TCID50 of the chimeric virus for expressing the extracellular domain of the TGEV S protein can be increased by 6.2 times compared with that of the chimeric PEDV for expressing the TGEV S protein. The recombinant virus is used as an inactivated vaccine for immunizing mice, so that the immunogenicity of TGEV S protein can be greatly improved, a body is stimulated to generate a higher-level neutralizing antibody aiming at TGEV, and the titer of the neutralizing antibody aiming at TGEV is improved by 16.8 times. Therefore, more possibilities are provided for modification of viral genomes, a novel method for improving immunogenicity of the TGEV inactivated vaccine is invented, and a basis is provided for research and development of the TGEV vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a chimeric PEDV infectious clone plasmid, a PEDV chimeric virus, and a construction method and application thereof. Background Art

[0002] Over the past two decades, with the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome virus (MERS-CoV), severe acute respiratory syndrome coronavirus type 2 (SARS-CoV 2), porcine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine delta coronavirus (PDCoV), we have come to realize that coronaviruses remain one of the greatest threats to human and livestock health.

[0003] Transmissible gastroenteritis (TGE) is a contagious viral diarrhea caused by transmissible gastroenteritis virus (TGEV). TGEV is a member of the alpha coronavirus and mainly infects the enterocytes of the small intestinal villi of pigs, causing symptoms such as diarrhea, vomiting, dehydration, and loss of appetite. The mortality rate of infected piglets can reach 100%, posing a huge threat and economic loss to the pig industry. Although the detection rate of TGEV in pig farms has been low in recent years, there are still epidemic strains, so we must not relax the prevention and control of TGEV. TGEV S protein has been proven to be involved in receptor recognition and membrane fusion processes, and plays an important role in the virulence of the virus, the tissue tropism of the host, the cell tropism, and the pathogenic mechanism of the virus. The S protein contains multiple antigenic epitopes with immune protection and is a key protein that stimulates the host to produce neutralizing antibodies. Therefore, the S protein is also a key target for vaccine design.

[0004] During the evolution of coronaviruses, recombination occurs not only between viruses of the same species, but also between viruses of different species. For example, researchers have detected recombinant viruses with the TGEV genome as the backbone and the PEDV S gene in both Italy and Germany. Researchers have also constructed an attenuated virus that expresses the PEDV S protein with the TGEV genome as the backbone, which can produce protection against PEDV after immunizing piglets. German researchers used the PEDV reverse genetics platform to replace the PEDV S protein with the TGEVS gene, saving rPEDV-TGEV S and confirming the recombination between PEDV and TGEV.

[0005] Vaccine immunization is one of the effective means of preventing and controlling TGEV. Inactivated vaccines have the advantage of high safety, so they have become the first choice of many farmers. However, the main problem faced by inactivated vaccines is low immunogenicity. Multiple immunizations are required to induce the body to produce an ideal level of neutralizing antibodies. Therefore, how to improve the immunogenicity of inactivated vaccines is of great significance. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a chimeric PEDV infectious clone plasmid and a construction method thereof.

[0007] The technical problem that the present invention needs to solve is to provide a PEDV chimeric virus and a rescue method thereof.

[0008] The technical problem that the present invention also aims to solve is to provide a chimeric PEDV infectious clone plasmid or the PEDV chimeric virus for use in preparing a TGEV vaccine or a TGEV antibody.

[0009] The technical problem that the present invention needs to solve is to provide a TGEV vaccine.

[0010] The final technical problem to be solved by the present invention is to provide a TGEV neutralizing antibody.

[0011] Technical solution: In order to solve the above technical problems, the present invention provides a chimeric PEDV infectious clone plasmid, which uses the PEDV infectious clone plasmid as a skeleton, replaces the TGEV S gene extracellular region with the PEDV S gene extracellular region, and the nucleotide sequence of the PEDV S gene extracellular region is shown in SEQ ID NO.1, and the nucleotide sequence of the TGEV S gene extracellular region is shown in SEQ ID NO.2.

[0012] Among them, the PEDV infectious clone plasmid is pBAC-PEDV GX4 / 2021, and the replacement methods include Red recombination method, enzyme cutting-ligation and CRISPR-Cas9-homologous recombination technology.

[0013] The present invention also includes a method for constructing the chimeric PEDV infectious clone plasmid, comprising the following steps:

[0014] (1) pBAC-PEDV GX4 / 2021 and cDNA of TGEV strain TGEV SHXB were used as templates to amplify the first part of the target fragment (2 fragments) and the second part of the target fragment (2 fragments), respectively, and then pET30a was used as a template to amplify the middle fragment.

[0015] (2) Amplification of the target fragment: the five fragments including the first part of the fragment (2 fragments), the second part of the fragment (2 fragments) and the middle fragment are connected to obtain the target fragment;

[0016] (3) The first step is Red recombination: the targeting fragment is transformed into competent cells and the positive clones are obtained by streaking purification and verification;

[0017] (4) The second step is Red recombination: positive clones are selected for temperature, L-arabinose induction, and antibiotic screening to further obtain the final positive clones.

[0018] Among them, the specific steps of step (1) are: using pBAC-PEDV GX4 / 2021 as a template, using primer pair PE-nsp16 F1 and PEDV-nsp16 R to amplify the PE-nsp16 fragment; using the cDNA of TGEV SHXB as a template, using primer pair TGEV-S-1F and TGEV-S-1R, amplifying the first half of the TGEV S gene extracellular region TGEV-S-1 fragment; using pET30a as a template, using primer pair I-SceI-CATpro F1 and CATpro-Kan R, amplifying fragment Kan; using the cDNA of TGEV SHXB as a template, using primer pair TGEV-S-2F and TGEV-S-TM R, amplifying the second half of the TGEV S gene extracellular region TGEV-S-2 fragment; using pBAC-PEDV GX4 / 2021 as a template, using primer pair PE-S-TM F and PE-Sc R, amplifying PEDV PE-S-TM fragment of the intracellular region of the S gene.

[0019] The specific steps of step (2) are as follows: using the fragment PE-nsp16, the fragment TGEV-S-1 and the fragment Kan as templates, using the primer pair PE-nsp16 F1 and CATpro-Kan R, and using overlap PCR to amplify the first half of the target fragment b-TGEV-S1-Kan; using the fragment Kan, the fragment TGEV-S-2 and the fragment PE-S-TM as templates, using the primer pair I-SceI-CATproF1 and PE-Sc R, amplifying the second half of the target fragment Kan-TGEV-S-2-PE-S-TM. Then, using the b-TGEV-S1-Kan fragment and the Kan-TGEV-S-2-PE-S-TM fragment as templates, using the primer pair PE-nsp16F1 and PE-Sc R, amplifying the target fragment b-TGEV-S-extrD-Kan-c;

[0020] Preferably, the specific steps of step (3) are: using electroporation to transform the target fragment b-TGEV-S-extrD-Kan-c into E.coli GS1783-pBAC-PEDV GX4 / 2021 competent cells, coating on an LB plate containing chloramphenicol and kanamycin resistance, culturing in an incubator for the first step of recombination, using primers PE-nsp16 F1 and PE-S-cR to perform colony PCR identification, streaking and purifying the correctly identified positive colonies on a plate containing kanamycin resistance, culturing, and again using primers PE-nsp16 F1 and PE-Sc R to perform colony PCR identification, and the purified positive colonies are named pBAC-PEDV-TGEVS-extrD-Kan;

[0021] Preferably, the specific steps of step (4) are: picking a positive clone and inoculating it in an LB liquid culture medium containing chloramphenicol resistance, shaking and culturing it on a shaker until it becomes turbid, adding an LB liquid culture medium containing L-arabinose and chloramphenicol resistance thereto, continuing to culture, culturing it on a shaker, and then transferring it to a shaker for further culture, taking the bacterial solution and applying it to an LB plate containing L-arabinose and chloramphenicol resistance, and after the bacterial solution is fully absorbed, inverting and culturing it in a bacterial incubator, picking the obtained single colony and spot-coating it on an LB plate containing chloramphenicol resistance and an LB plate containing kanamycin resistance, respectively, the colony that does not grow in the kanamycin resistance LB solid culture medium and grows in the chloramphenicol LB solid culture medium is the colony with successful recombination, and the obtained positive clone is named pBAC-PEDV-TGEV S-extrD.

[0022] The present invention also includes a PEDV chimeric virus, which is obtained by rescuing cells after transfecting the chimeric PEDV infectious clone plasmid or the infectious clone plasmid obtained by the construction method.

[0023] The present invention also includes a method for rescuing a PEDV chimeric virus, comprising the following steps: co-transfecting cells with the chimeric PEDV infectious clone plasmid or the infectious clone plasmid obtained by the construction method and a helper plasmid.

[0024] The present invention also includes the use of the chimeric PEDV infectious clone plasmid or the PEDV chimeric virus in the preparation of PEDV vaccine or PEDV antibody.

[0025] The present invention also includes a PEDV vaccine, which contains the chimeric PEDV infectious clone plasmid or the PEDV chimeric virus.

[0026] The present invention also includes a PEDV neutralizing antibody, which is obtained by infecting animals with the PEDV chimeric virus.

[0027] The present invention is the first to insert the TGEV S protein extracellular region (shown in SEQ ID NO.2), the PDCoV S protein extracellular region (shown in SEQID NO.3) and the SADS-CoV S protein extracellular region (shown in SEQ ID NO.4) into the PEDV genome. After many attempts, only the chimeric virus rPEDV-TGEV S-extrD expressing the TGEV S protein extracellular region was successfully rescued, indicating that using the PEDV genome as the skeleton, even if it is difficult to rescue the S extracellular region expressing other porcine coronaviruses, it also provides more possibilities for the modification of the viral genome.

[0028] Beneficial effects: For the first time, the present invention uses the PEDV genome as a skeleton to express the extracellular region of the TGEV S protein and successfully rescues the recombinant virus. Immunizing mice with this recombinant virus as an inactivated vaccine greatly improves the immunogenicity of the TGEV S protein, stimulates the body to produce higher levels of neutralizing antibodies against TGEV, and increases the titer of neutralizing antibodies against TGEV by 16.8 times. Therefore, this study provides more possibilities for the modification of the viral genome, and also invents a new method to improve the immunogenicity of the TGEV inactivated vaccine, providing a basis for the research and development of the TGEV vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram for the construction of PEDV chimeric virus;

[0030] Figure 2 PCR identification of recombinant plasmids;

[0031] Figure 3 Identification of chimeric viruses; (A) IFA identification of chimeric viruses; (B) Western blot identification of chimeric viruses; (C) TCID of chimeric viruses 50 Determination of

[0032] Figure 4 For the determination of neutralizing activity of mouse serum. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods.

[0034] 1. Experimental Materials

[0035] PDCoV virus strain PDCoV GX2022-1 (Gebank accession number: OQ736717), SADS-CoV virus strain SADS-CoV-GD-1 (Gebank accession number: PQ000331), TGEV strain PEDV SHXB (GenBank accession number: KP202848), pET30a, Vero cells, E. coli GS1783-pBAC-PEDV GX4 / 2021 strain, pBAC-PEDV GX4 / 2021, and pCAGGS-PEDV N are stored in this laboratory. Anti-PEDV-N monoclonal antibodies were prepared and stored in this laboratory. The above plasmids, strains, and monoclonal antibodies were published in China Patent Publication No. CN116445528A Construction method of recombinant porcine epidemic diarrhea virus infectious clone and its infectious clone and application. Goat anti-mouse IgG (H+L)-HRP and anti-Actin monoclonal antibodies were purchased from Wuhan ABclonal Company; ECL luminescent liquid was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd. 6-8 weeks old BALB / c female mice were purchased from the Experimental Animal Center of the Comparative Medical College of Yangzhou University.

[0036] 2. Primer design

[0037] Primers were designed according to the nucleotide sequences of pBAC-PEDV GX4 / 2021, PDCoV S protein (GenBank accession number: OQ736717, position: 19276-22755), SADS-CoV S protein (GenBank accession number: PQ000331, position: 20486-23878), and TGEV S protein (GenBank accession number: KP202848, position: 20365-24708) ( Table 1 ), and the primers were synthesized by Nanjing Qingke Biological Co., Ltd.

[0038] Table 1 Primer usage for recombinant plasmid construction

[0039]

[0040]

[0041] 3. Construction of chimeric PEDV infectious clone plasmids expressing other porcine coronavirus S proteins

[0042] According to the schematic diagram of recombinant plasmid construction ( Figure 1 ), using Red recombination technology to construct chimeric PEDV infectious clone plasmids expressing TGEV S protein, PDCoV S protein, and SADS-CoV S protein. The specific construction process is as follows:

[0043] 3.1 Construction of chimeric PEDV infectious clone plasmid expressing TGEV S protein

[0044] (1) Amplification of DNA fragments. Using pBAC-PEDV GX4 / 2021 as a template, the primer pair PE-nsp16F1 and PEDV-nsp16 R were used to amplify the PE-nsp16 fragment; using the cDNA of the TGEV strain TGEV SHXB as a template, the primer pair TGEV-S-1F and TGEV-S-1R were used to amplify the first half of the TGEV S gene, the TGEV-S-1 fragment; using pET30a as a template, the primer pair I-SceI-CATpro F1 and CATpro-Kan R were used to amplify the fragment Kan; using the cDNA of TGEV SHXB as a template, the primer pair TGEV-S-2F and TGEV-S-2R were used to amplify the second half of the TGEV S gene, the TGEV-S-2 fragment; using pBAC-PEDV GX4 / 2021 as a template, the primer pair PE-S F2 and PE-Sc R were used to amplify the 3' end region PE-S-2 fragment of the PEDV S gene. The PCR reaction system is shown in Table 2, and the PCR amplification conditions are shown in Table 3.

[0045] Table 2 PCR reaction system

[0046] <![CDATA[ddH2O]]> 22μL Upstream primer 1μL Downstream primer 1μL template 1μL PrimeSTAR Max Premix (2X) 25μL

[0047] Table 3 PCR amplification conditions

[0048]

[0049] (2) Amplification of the target fragment b-TGEV-S-Kan-c. According to the PCR reaction system in Table 4, the fragment PE-nsp16, the fragment TGEV-S-1 and the fragment Kan were used as templates 1, template 2 and template 3, and the primer pair PE-nsp16 F1 and CATpro-KanR were used to amplify the first half of the target fragment b-TGEV-S1-Kan by overlap PCR. The fragment Kan, the fragment TGEV-S-2 and the fragment PE-S-2 were used as templates 1, template 2 and template 3, and the primer pair I-SceI-CATpro F1 and PE-Sc R were used to amplify the second half of the target fragment Kan-TGEV-S-2-PE-S-2 by overlap PCR. According to Table 5, the b-TGEV-S1-Kan fragment and the Kan-TGEV-S-2-PE-S-2 fragment were used as templates 4 and 5, and the primer pair PE-nsp16 F1 and PE-Sc R were used to amplify the target fragment b-TGEV-S-Kan-c. All PCR reaction processes were performed according to the conditions in Table 3 for PCR amplification.

[0050] Table 4 Overlap PCR reaction system

[0051] <![CDATA[ddH2O]]> Make up to 50 μL Upstream primer 1μL Downstream primer 1μL Template 1 1μL Template 2 1μL Template 3 1μL PrimeSTAR Max Premix (2X) 25μL

[0052] Table 5 Overlap PCR reaction system

[0053] <![CDATA[ddH2O]]> Make up to 50 μL Upstream primer 1μL Downstream primer 1μL Template 4 1μL Template 5 1μL PrimeSTAR Max Premix (2X) 25μL

[0054] (3) Electroporation of the target fragment for the first step of Red recombination. The target fragment b-TGEV-S-Kan-c was transformed into E. coli GS1783-pBAC-PEDV GX4 / 2021 competent cells by electroporation, spread on LB plates containing chloramphenicol and Kan resistance, and cultured in a 32°C incubator for 24 h for the first step of recombination.

[0055] (4) Pick a single colony on the plate and perform colony PCR identification using primers PE-nsp16 F1 and PE-Sc R. The positive colony that has been identified correctly is streaked and purified on a plate containing Kaner resistance and incubated at 32°C for 16 h. Colony PCR identification is performed again using primers PE-nsp16 F1 and PE-Sc R, and the purified positive colony is named pBAC-PEDV-TGEV S-Kan.

[0056] (5) Step 2: Red recombination. Pick a positive single colony and inoculate it into 2 mL of LB liquid medium containing chloramphenicol resistance. Incubate it in a 32°C shaker until it becomes turbid. Add 2 mL of LB liquid medium containing 2% L-arabinose and chloramphenicol resistance. Continue to incubate it for 1 hour. Then, incubate it in a 42°C shaker for 30 minutes. Then, transfer it to a 32°C shaker and continue to incubate it for 3 hours.

[0057] (6) Take 100 μL of bacterial solution and do 10 4 Dilution was performed by 200 μL dilution, and 200 μL of the diluted bacterial solution was spread on an LB plate containing 1% L-arabinose and chloramphenicol resistance. After the bacterial solution was fully absorbed, it was inverted and cultured in a 32°C bacterial incubator for about 24 h.

[0058] (7) Pick the single colonies obtained in step (6) and apply them to LB plates containing chloramphenicol resistance and kanamycin resistance, respectively. The colonies that do not grow in the kanamycin resistance LB solid medium and grow in the chloramphenicol LB solid medium are the colonies with successful recombination, and the plasmid is identified by PCR reaction, and the obtained positive clone is named pBAC-PEDV-TGEV S.

[0059] 3.2 Construction of chimeric PEDV infectious clone plasmid expressing PDCoV S protein

[0060] (1) Amplification of DNA fragments. According to the PCR reaction system in Table 2, the cDNA of PDCoV GX2022-1 was used as a template, and the primer pair PDCoV-S-1F and PDCoV-S-1R was used to amplify the first half of the PDCoV S gene, the PDCoV-S-1 fragment, and the primer pair PDCoV-S-2F and PDCoV-S-2R was used to amplify the second half of the PDCoV S gene, the PDCoV-S-2 fragment. PCR amplification was performed according to the conditions in Table 3.

[0061] (2) Amplification of the target fragment b-PDCoV-S-Kan-c. According to the PCR reaction system in Table 4, the fragment PE-nsp16, the fragment PDCoV-S-1 and the fragment Kan were used as templates 1, template 2 and template 3, and the primer pair PE-nsp16F1 and CATpro-KanR were used to amplify the first half of the target fragment b-PDCoV-S1-Kan by overlap PCR. The fragment Kan, the fragment PDCoV-S-2 and the fragment PE-S-2 were used as templates 1, template 2 and template 3, and the primer pair I-SceI-CATpro F1 and PE-Sc R were used to amplify the second half of the target fragment Kan-PDCoV-S-2-PE-S-2. According to Table 5, the b-PDCoV-S1-Kan fragment and the Kan-PDCoV-S-2-PE-S-2 fragment were used as templates 4 and 5, and the primer pair PE-nsp16 F1 and PE-Sc R were used to amplify the target fragment b-PDCoV-S-Kan-c. The above PCR reactions were all performed according to the PCR reaction conditions in Table 3.

[0062] (3) Electroporation of the target fragment, the first step of Red recombination, and streak purification of positive colonies. This step is the same as step 3.1 (3) (4), the target fragment is b-PDCoV-S-Kan-c, and the obtained positive plasmid is named pBAC-PEDV-PDCoV S-Kan.

[0063] (4) The second step is Red recombination and screening of positive colonies. This step is the same as step 3.1 (5)(6)(7). The obtained positive clone is named pBAC-PEDV-PDCoV S.

[0064] 3.3 Construction of chimeric PEDV infectious clone plasmid expressing SADS-CoV S protein

[0065] (1) Amplification of DNA fragments. According to Table 2, using the cDNA of SADS-CoV-GD-1 as a template, the primer pair SADS-S-1F and SADS-S-1R were used to amplify the first half of the SADS-CoV S gene, the SADS-S-1 fragment, and the primer pair SADS-S-2F and SADS-S-2R were used to amplify the second half of the SADS-CoV S gene, the SADS-S-2 fragment. PCR amplification was performed according to the PCR reaction system in Table 3.

[0066] (2) Amplification of the target fragment b-SADS-S-Kan-c. According to Table 4, the fragment PE-nsp16, the fragment SADS-S-1 and the fragment Kan were used as templates 1, template 2 and template 3, and the primer pair PE-nsp16 F1 and CATpro-Kan R were used to amplify the first half of the target fragment b-SADS-S1-Kan by overlapPCR. The fragment Kan, the fragment SADS-S-2 and the fragment PE-S-2 were used as templates 1, template 2 and template 3, and the primer pair I-SceI-CATpro F1 and PE-S-cR were used to amplify the second half of the target fragment Kan-SADS-S-2-PE-S-2. According to Table 5, the b-SADS-S1-Kan fragment and the Kan-SADS-S-2-PE-S-2 fragment were used as templates 4 and 5, and the primer pair PE-nsp16 F1 and PE-S-cR were used to amplify the target fragment b-SADS-S-Kan-c. The above PCR reactions were all performed according to the PCR reaction conditions in Table 3.

[0067] (3) Electroporation of the target fragment, the first step of Red recombination, and streak purification of positive colonies. This step is the same as step 3.1 (3) (4), the target fragment is b-SADS-S-Kan-c, and the obtained positive plasmid is named pBAC-PEDV-SADS-CoVS-Kan.

[0068] (4) The second step is Red recombination and screening of positive colonies. This step is the same as step 3.1 (5)(6)(7). The obtained positive clone is named pBAC-PEDV-SADS-CoV S.

[0069] 4. Construction of chimeric PEDV infectious clone plasmids expressing the extracellular region of other porcine coronavirus S proteins

[0070] In order to explore whether the intracellular region of the S protein affects the rescue and growth characteristics of the chimeric virus, according to the schematic diagram of the recombinant plasmid construction ( Figure 1), Red recombination technology was used to construct chimeric PEDV infectious clone plasmids expressing the TGEV S protein extracellular region, the PDCoV S protein extracellular region, and the SADS-CoV S protein extracellular region. The specific construction process is as follows:

[0071] 4.1 Construction of chimeric PEDV infectious clone plasmid expressing the extracellular region of TGEV S protein

[0072] (1) Amplification of DNA fragments. Using the cDNA of TGEV SHXB as a template, the primer pair TGEV-S-2F and TGEV-S-TM R was used to amplify the TGEV-S-2' fragment of the second half of the extracellular region of the TGEV S gene; using pBAC-PEDV GX4 / 2021 as a template, the primer pair PE-S-TM F and PE-Sc R was used to amplify the PE-S-TM fragment of the extracellular region of the PEDV S gene. The PCR reaction system is shown in Table 2, and the PCR amplification conditions are shown in Table 3.

[0073] (2) Amplification of the target fragment b-TGEV-S-extrD-Kan-c. According to the PCR reaction system in Table 4, fragment Kan, fragment TGEV-S-2' and fragment PE-S-TM were used as templates 1, template 2 and template 3, and primer pairs I-SceI-CATpro F1 and PE-Sc R were used to amplify the second half of the target fragment Kan-TGEV-S-2-PE-S-TM by overlap PCR. According to the PCR reaction system in Table 4, b-TGEV-S1-Kan fragment and Kan-TGEV-S-2-PE-S-TM fragment were used as templates 4 and template 5, and primer pairs PE-nsp16 F1 and PE-Sc R were used to amplify the target fragment b-TGEV-S-extrD-Kan-c. All PCR reaction processes were performed according to the conditions of Table 3 for PCR amplification.

[0074] (3) Electroporation of the target fragment, the first step of Red recombination, and streak purification of positive colonies. This step is the same as step 3.1 (3) (4), the target fragment is b-TGEV-S-extrD-Kan-c, and the obtained positive plasmid is named pBAC-PEDV-TGEVS-extrD-Kan.

[0075] (4) The second step is Red recombination and screening of positive colonies. This step is the same as step 3.1 (5), (6), (7). The obtained positive clone is named pBAC-PEDV-TGEV S-extrD.

[0076] 4.2 Construction of chimeric PEDV infectious clone plasmid expressing the extracellular region of PDCoV S protein

[0077] (1) Amplification of DNA fragments. According to the PCR reaction system in Table 2, the cDNA of PDCoV GX2022-1 was used as a template, and the primer pair PDCoV-S-2F and PDCoV-S-TM R were used to amplify the second half of the extracellular region of the PDCoV S gene, the PDCoV-S-2' fragment, according to the reaction conditions in Table 3.

[0078] (2) Amplification of the target fragment b-PDCoV-S-extrD-Kan-c. According to the PCR reaction system in Table 4, the fragment Kan, the fragment PDCoV-S-2' and the fragment PE-S-TM were used as templates 1, template 2 and template 3, and the primer pair I-SceI-CATproF1 and PE-Sc R were used to amplify the second half of the target fragment Kan-PDCoV-S-2-PE-S-TM. According to Table 5, the b-PDCoV-S1-Kan fragment and the Kan-PDCoV-S-2-PE-S-TM fragment were used as templates 4 and template 5, and the primer pair PE-nsp16 F1 and PE-Sc R were used to amplify the target fragment b-PDCoV-S-extrD-Kan-c. The above PCR reactions were all performed according to the PCR reaction conditions in Table 3 for PCR amplification.

[0079] (3) Electroporation of the target fragment, the first step of Red recombination, and streak purification of positive colonies. This step is the same as step 3.1 (3) (4), the target fragment is b-PDCoV-S-extrD-Kan-c, and the obtained positive plasmid is named pBAC-PEDV-PDCoV S-extrD-Kan.

[0080] (4) The second step is Red recombination and screening of positive colonies. This step is the same as step 3.1 (5), (6), (7). The obtained positive clone is named pBAC-PEDV-PDCoV S-extrD.

[0081] 4.3 Construction of chimeric PEDV infectious clone plasmid expressing the extracellular region of SADS-CoV S protein

[0082] (1) Amplification of DNA fragments. According to Table 2, the cDNA of SADS-CoV-GD-1 was used as a template, and the primer pair SADS-S-2F and SADS-S-TM R were used to amplify the SADS-S-2' fragment, the second half of the extracellular region of the SADS-CoV S gene, according to the PCR reaction conditions in Table 3.

[0083] (2) Amplification of the target fragment b-SADS-S-extrD-Kan-c. According to Table 4, the fragment Kan, the fragment SADS-S-2 and the fragment PE-S-TM were used as templates 1, template 2 and template 3, and the primer pair I-SceI-CATpro F1 and PE-Sc R were used to amplify the second half of the target fragment Kan-SADS-S-2-PE-S-TM. According to Table 5, the b-SADS-S1-Kan fragment and the Kan-SADS-S-2-PE-S-TM fragment were used as templates 4 and template 5, and the primer pair PE-nsp16 F1 and PE-Sc R were used to amplify the target fragment b-SADS-S-extrD-Kan-c. The above PCR reactions were all performed according to the PCR reaction conditions in Table 3 for PCR amplification.

[0084] (3) Electroporation of the target fragment, the first step of Red recombination, and streak purification of positive colonies. This step is the same as step 3.1 (3) (4), the target fragment is b-SADS-S-extrD-Kan-c, and the obtained positive plasmid is named pBAC-PEDV-SADS-CoV S-extrD-Kan.

[0085] (4) The second step is Red recombination and screening of positive colonies. This step is the same as step 3.1 (5), (6), (7). The obtained positive clone is named pBAC-PEDV-SADS-CoV S-extrD.

[0086] like Figure 2 The results of nucleic acid electrophoresis showed that DNA fragments of the size of the S gene of the corresponding virus could be amplified using the recombinant plasmid as a template, indicating that the infectious clone plasmids of the six chimeric viruses were successfully constructed.

[0087] 5. Rescue of recombinant viruses

[0088] (1) After the positive colonies of pBAC-PEDV-TGEV S, pBAC-PEDV-PDCoV S, pBAC-PEDV-SADS-CoV S, pBAC-PEDV-TGEV S-extrD, pBAC-PEDV-PDCoV S-extrD or pBAC-PEDV-SADS-CoV S-extrD were expanded and cultured, the plasmids were extracted using a plasmid extraction kit.

[0089] (2) Evenly spread ST-R cells in good growth state into 6-well plates. When the cell density reaches 70%-80%, Transfection was performed according to the instructions of 3000 reagent.

[0090] (3) The amount of plasmids used in each well is as follows: 0.2 μg of auxiliary plasmid pCAGGS-PEDV-N, 1.8 μg of recombinant virus infectious clone plasmid (pBAC-PEDV-TGEV S, pBAC-PEDV-PDCoV S, pBAC-PEDV-SADS-CoV S, pBAC-PEDV-TGEV S-extrD, BAC-PEDV-PDCoV S-extrD or pBAC-PEDV-SADS-CoV S-extrD). The wild-type PEDV infectious clone plasmid (pBAC-PEDV GX4 / 2021) was used as a positive control. 4-6 hours after transfection, the cell culture medium was replaced with fresh DMEM medium containing 10% FBS, and 24 hours after transfection, the cell culture supernatant was discarded, the cell monolayer was washed twice with sterilized PBS buffer (0.01M, pH=7.2), 2 mL of DMEM medium containing 2% FBS was added to the cells transfected with pBAC-PEDV-TGEV S or pBAC-PEDV-TGEV S-extrD, and 2 mL of DMEM medium containing 2 μg / mL trypsin was added to the cells transfected with pBAC-PEDV-PDCoV S, pBAC-PEDV-SADS-CoV S, pBAC-PEDV-PDCoV S-extrD or pBAC-PEDV-SADS-CoV S-extrD. After 3 days, the cell supernatant was collected and fresh ST cells were infected again.

[0091] (4) When the cells become round, swollen, and have nuclear and cytoplasmic lesions, the recombinant virus is successfully rescued. The rescued viruses are named rPEDV-TGEV S, rPEDV-PDCoV S, rPEDV-SADS-CoV S, rPEDV-TGEV S-extrD, rPEDV-PDCoV S-extrD, and rPEDV-SADS-CoV S-extrD. The rescued viruses are serially passaged in ST cells, and each generation of virus fluid is labeled and stored at -80°C.

[0092] After continuous passage, cells infected with only the chimeric viruses rPEDV-TGEV S expressing the full-length S protein of TGEV and rPEDV-TGEV S-extrD expressing the extracellular region of TGEV S were observed to become round and swollen, indicating that the chimeric viruses rPEDV-TGEV S and rPEDV-TGEV S-extrD were successfully rescued. During the continuous passage of the chimeric viruses rPEDV-PDCoV S expressing the full-length S protein of PDCoV, rPEDV-PDCoV S-extrD expressing the extracellular region of PDCoV S protein, rPEDV-SADS-CoV S expressing the full-length S protein of SADS-CoV, and rPEDV-SADS-CoV S-extrD expressing the extracellular region of SADS-CoV S protein, no characteristic cytopathic changes such as rounding, swelling, and nucleosomes were observed in the cells. After multiple rescues and virus passages, cells and supernatants were collected and tested by quantitative PCR for viral RNA. As shown in Table 6, RNA of the PEDV N gene was detected in cells and supernatants infected with the chimeric viruses rPEDV-TGEV S and rPEDV-TGEV S-extrD, while no RNA of the PEDV N gene was detected in cells and supernatants infected with the other four chimeric viruses, indicating that the rescue of rPEDV-PDCoV S, rPEDV-SADS-CoV S, rPEDV-PDCoV S-extrD and rPEDV-SADS-CoV S-extrD failed.

[0093] Table 6 Quantitative PCR detection of chimeric virus rescue

[0094]

[0095] 6. Identification of chimeric viruses

[0096] 6.1 Indirect immunofluorescence identification

[0097] The rescued viruses rPEDV-TGEV S and rPEDV-TGEV S-extrD were used to infect ST cells for 36 hours, and then the cells were fixed and detected using a monoclonal antibody against the PEDV-N protein. The specific steps are as follows:

[0098] (1) ST 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 P0 generation rescue virus solution was inoculated into the cell monolayer, and a well without virus solution was set. After incubation in a 37°C, 5% CO2 constant temperature incubator for 2 h, the supernatant was discarded, 1 mL of DMEM medium containing 2% FBS was added, and the culture was continued for 36 h. The cell culture supernatant was collected and marked as P1 generation.

[0099] (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.

[0100] (3) Add pre-cooled PBS solution containing 0.1% TritonX-100 and 2% BSA, permeabilize and block the membrane at room temperature for 30 minutes, and wash twice with PBS.

[0101] (4) Add 1:2000 diluted anti-PEDV N monoclonal antibody, incubate at 37°C for 1 h, and wash three times with PBS.

[0102] (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.

[0103] (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.

[0104] like Figure 3 As shown in A, after infection with rPEDV-TGEV S and rPEDV-TGEV S-extrD, ST had green fluorescence, while the negative control wells had no green fluorescence, indicating that we successfully rescued the chimeric virus rPEDV-TGEV S expressing the TGEV S protein and the chimeric virus rPEDV-TGEV S-extrD expressing the extracellular region of the TGEV S protein.

[0105] 6.2 Western blot identification of chimeric viruses

[0106] (1) ST cells were plated in a 12-well plate in advance until the cell density reached 90%.

[0107] (2) rPEDV-TGEV S and rPEDV-TGEV S-extrD were used to infect ST cells at an MOI of 0.01 for 24 h. The virus-uninfected group was set as a negative control.

[0108] (3) Collect the supernatant, wash the cells twice with PBS buffer, add 150 μL of RIPA lysis buffer to each well, and collect the protein sample.

[0109] (4) The collected protein samples were subjected to western blot identification, using anti-PEDV N protein monoclonal antibody as the primary antibody and goat anti-mouse IgG (H+L)-HRP as the secondary antibody.

[0110] The results showed that N protein expression could be detected in the virus-infected group ( Figure 3 B), showing that we successfully rescued the chimeric viruses rPEDV-TGEV S and rPEDV-TGEV S-extrD.

[0111] 6.3 TCID of chimeric viruses 50 Determination

[0112] The supernatant collected in step 6.2(2) was used for chimeric virus TCID 50 The specific steps for determination are as follows:

[0113] (1) One day in advance, ST cells were plated into 96-well plates and infected with viruses when the density was greater than 90%.

[0114] (2) Prepare 10 sterile 1.5 mL centrifuge tubes and add 900 μL of DMEM medium containing 2% FBS to each tube. Add 100 μL of the virus stock solution to the first centrifuge tube and mix thoroughly. Then take 100 μL and add it to the second tube and mix thoroughly. Repeat this process to make 10-fold gradient dilutions.

[0115] (3) Remove the 96-well plate from the cell culture incubator, discard the culture medium in the wells, wash the cells twice with PBS, and add the diluted virus solution to the 96-well plate in sequence, 100 μL per well. Make 4 parallels for each dilution. Add 2% FBS DMEM culture medium to the negative control wells and continue culturing in a 37°C incubator with 5% CO2.

[0116] (4) After 2 days of culture, observe the cytopathic effect, count the CPE wells, and calculate the virus titer according to the Karber method.

[0117] like Figure 3 As shown in C, the titer of rPEDV-TGEV S-extrD is 6.2 times higher than that of rPEDV-TGEV S, indicating that after the combination of the extracellular region of the TGEV S protein and the intracellular region of the PEDV S protein, the chimeric virus has better growth characteristics.

[0118] 7. Animal testing

[0119] 7.1 Immunization of mice

[0120] In order to explore the difference in immunogenicity between the recombinant virus expressing the extracellular region of the TGEV S protein using the PEDV genome as a backbone and the wild-type TGEV strain, mice were immunized according to the experimental scheme in Table 7. The DMEM immunization group was used as a negative control. Ten days after the third immunization, mouse sera were collected to detect the neutralizing antibody titer.

[0121] Table 7 Mouse immunization program

[0122]

[0123]

[0124] 7.2 Determination of neutralizing antibodies in mouse serum

[0125] (1) Ten days after the third immunization of mice, about 900 μL of blood was collected from the retro-orbital vein of the mice using a glass capillary pipette and placed in a 37°C constant temperature incubator for 1 h to allow serum to precipitate. The blood was centrifuged at 3000 × g and 4°C for 10 min, and the upper layer of serum was collected, aliquoted, and stored in a -80°C refrigerator for testing.

[0126] (2) ST cells were plated into a 96-well plate one day in advance, and the experiment was performed when the cell density reached 90%.

[0127] (3) The serum after the third immunization was inactivated at 56°C for 30 min and filtered using a 0.22 μm filter in a clean bench.

[0128] (4) The serum was diluted 2-fold with DMEM medium. 250 μL of diluted antibody and 250 μL of diluted TGEV strain TGEV SHXB virus solution (100 TCID 50 / 100 μL) and mix thoroughly, and incubate at 37°C for 1 h.

[0129] (5) After the incubation, discard the culture medium in the 96-well plate, take 100 μL of serum-virus mixture and add it 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 incubated in a 5% CO2, 37°C incubator for 1 h.

[0130] (6) After the reaction, discard the supernatant, wash twice with PBS, add 100 μL of DMEM medium containing 2% FBS to the cell wells, continue to culture for 72 hours, observe the cytopathic effect, and calculate the neutralizing titer of the immunized mouse serum. The calculation formula is: lg (neutralizing antibody titer) = L + d (S-0.5). (L: the logarithm of the highest serum dilution; d: group distance, that is, dilution coefficient, which is -0.3 in a 2-fold serial dilution; S: the sum of the ratios of the number of CPE wells in each group to the number of inoculated wells.) Figure 4 As shown in the figure, compared with the negative control group, mice immunized with TGEV SHXB could hardly produce neutralizing antibodies against TGEV, while mice immunized with rPEDV-TGEV S-extrD could stimulate mice to produce higher levels of neutralizing antibodies, with a neutralizing antibody titer of about 2. 6 Compared with the TGEV wild-type strain TGEV SHXB immunization group, using rPEDV-TGEV S-extrD as an immunogen greatly enhanced the host's ability to produce neutralizing antibodies, and the neutralizing antibody titer increased by 16.8 times.

[0131] PEDV S gene extracellular region: SEQ ID NO.1

[0132]

[0133]

[0134] TGEV S gene extracellular region: SEQ ID NO.2

[0135]

[0136]

[0137] PDCoV S gene extracellular region: SEQ ID NO.3

[0138]

[0139]

[0140] SADS-CoV S gene extracellular region: SEQ ID NO.4

[0141]

[0142]

Claims

1. A chimeric PEDV infectious clone plasmid, characterized in that: The chimeric PEDV infectious clone plasmid uses the PEDV infectious clone plasmid as a skeleton, and replaces the PEDV S gene extracellular region with the TGEV S gene extracellular region. The nucleotide sequence of the PEDV S gene extracellular region is shown in SEQ ID NO.1, and the nucleotide sequence of the TGEV S gene extracellular region is shown in SEQ ID NO.

2.

2. The chimeric PEDV infectious clone plasmid according to claim 1, characterized in that: The PEDV infectious clone plasmid is pBAC-PEDV GX4 / 2021, and the replacement method includes Red recombination method.

3. The method for constructing the chimeric PEDV infectious clone plasmid according to claim 1 or 2, characterized in that: The following steps are involved: (1) pBAC-PEDV GX4 / 2021 and cDNA of TGEV strain TGEV SHXB were used as templates to amplify the first part of the target fragment and the second part of the target fragment, respectively, and then pET30a was used as a template to amplify the middle fragment; (2) Amplification of the target fragment: connecting the first part of the fragment, the second part of the fragment and the middle fragment obtained by the above amplification to obtain the target fragment; (3) The first step is Red recombination: the targeting fragment is transformed into competent cells and the positive clones are obtained by streaking purification and verification; (4) The second step is Red recombination: positive clones are selected for temperature, L-arabinose induction, and antibiotic screening to further obtain the final positive clones.

4. The method for constructing a chimeric PEDV infectious clone plasmid according to claim 3, characterized in that: The specific steps of step (1) are as follows: using pBAC-PEDV GX4 / 2021 as a template, using primer pair PE-nsp16 F1 and PEDV-nsp16 R, amplifying the PE-nsp16 fragment; using the cDNA of TGEV SHXB as a template, using primer pair TGEV-S-1F and TGEV-S-1R, amplifying the first half of the TGEV S gene extracellular region TGEV-S-1 fragment; using pET30a as a template, using primer pair I-SceI-CATproF1 and CATpro-Kan R, amplifying fragment Kan; using the cDNA of TGEV SHXB as a template, using primer pair TGEV-S-2F and TGEV-S-TM R, amplifying the second half of the TGEV S gene extracellular region TGEV-S-2 fragment; using pBAC-PEDV GX4 / 2021 as a template, using primer pair PE-S-TM F and PE-Sc R, amplifying PEDV PE-S-TM fragment of the intracellular region of the S gene.

5. The method for constructing a chimeric PEDV infectious clone plasmid according to claim 3, characterized in that: The specific steps of step (2) are as follows: using the fragment PE-nsp16, the fragment TGEV-S-1 and the fragment Kan as templates, using the primer pair PE-nsp16 F1 and CATpro-Kan R, and using overlap PCR to amplify the first half of the target fragment b-TGEV-S1-Kan; using the fragment Kan, the fragment TGEV-S-2 and the fragment PE-S-TM as templates, using the primer pair I-SceI-CATpro F1 and PE-Sc R, amplifying the second half of the target fragment Kan-TGEV-S-2-PE-S-TM. Then, using the b-TGEV-S1-Kan fragment and the Kan-TGEV-S-2-PE-S-TM fragment as templates, using the primer pair PE-nsp16 F1 and PE-Sc R, amplifying the target fragment b-TGEV-S-extrD-Kan-c; Preferably, the specific steps of step (3) are: using electroporation to transform the target fragment b-TGEV-S-extrD-Kan-c into E.coli GS1783-pBAC-PEDV GX4 / 2021 competent cells, coating on an LB plate containing chloramphenicol and kanamycin resistance, culturing in an incubator for the first step of recombination, using primers PE-nsp16 F1 and PE-S-cR to perform colony PCR identification, streaking and purifying the correctly identified positive colonies on a plate containing kanamycin resistance, culturing, and again using primers PE-nsp16 F1 and PE-Sc R to perform colony PCR identification, and the purified positive colonies are named pBAC-PEDV-TGEV S-extrD-Kan; Preferably, the specific steps of step (4) are: picking positive clones and inoculating them into LB liquid culture medium containing chloramphenicol resistance, shaking and culturing them on a shaker until turbidity, adding LB liquid culture medium containing L-arabinose and chloramphenicol resistance thereto, and then transferring them to a shaker for further culturing, applying the bacterial solution to an LB plate containing L-arabinose and chloramphenicol resistance, and inverting and culturing them in a bacterial incubator after the bacterial solution is fully absorbed, picking the obtained single colonies and spot-coating them on LB plates containing chloramphenicol resistance and LB plates containing kanamycin resistance, respectively, the colonies that do not grow in kanamycin resistance LB solid culture medium and grow in chloramphenicol LB solid culture medium are colonies with successful recombination, and the obtained positive clones are named pBAC-PEDV-TGEV S-extrD.

6. A PEDV chimeric virus, characterized in that The chimeric virus is obtained by transfecting cells with the chimeric PEDV infectious clone plasmid described in claim 1 or 2 or the infectious clone plasmid obtained by any construction method of claim 4 or 5 and then rescuing the cells.

7. A method for rescuing a PEDV chimeric virus, characterized in that: The method comprises the following steps: co-transfecting cells with the chimeric PEDV infectious clone plasmid described in claim 1 or 2 or the infectious clone plasmid obtained by the construction method in any one of claims 4 or 5 and a helper plasmid.

8. Use of the chimeric PEDV infectious clone plasmid according to claim 1 or 2 or the PEDV chimeric virus according to claim 6 in the preparation of TGEV vaccine or TGEV antibody.

9. A TGEV vaccine, characterized in that It contains the chimeric PEDV infectious clone plasmid according to claim 1 or the PEDV chimeric virus according to claim 6.

10. A TGEV neutralizing antibody, characterized in that The neutralizing antibodies are obtained by infecting animals with the PEDV chimeric virus of claim 6.

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