A method and application of swine D-coronavirus infectious cDNA clone and construction

Abstract

The present invention provides a swine delta coronavirus infectious cDNA clone and a construction method and application, and belongs to the field of biotechnology. The present invention provides a construction method for a swine delta coronavirus infectious cDNA clone, which divides the genome of the swine delta coronavirus into several fragments, and completes the cloning construction based on a one-step method of yeast homologous recombination, which has the characteristics of high efficiency, rapidity, and stability. The infectious cDNA clone of the present invention has good stability, and the swine delta coronavirus genome can be directly modified at the genetic level using CRISPR / Cas9 gene editing. The infectious cDNA clone provided by the present invention can directly rescue the recombinant virus by transfecting mammalian cells, simplifying the virus rescue operation process. The present invention also provides a construction method for a swine delta coronavirus infectious clone that can express HiBiT tag protein, which provides a good technical means for in-depth research on the infection mechanism of swine delta coronavirus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a swine delta coronavirus infectious cDNA clone and construction method and application. Background Art

[0002] Porcine deltacoronavirus (PDCoV) is a new porcine enteric coronavirus that has emerged in recent years. It mainly infects newborn piglets, causing diarrhea, vomiting, dehydration and even death in piglets. Since the outbreak in the United States in 2014, PDCoV has become widespread in pig herds in countries around the world, causing economic losses to the pig farming industry. As early as 2018, studies reported that PDCoV can infect human cells. Not only that, in December 2021, Nature magazine published a case report on three Haitian children infected with PDCoV, which makes PDCoV a pathogen with potential zoonotic risks and poses a threat to global public health. However, we still don’t know much about the basic scientific issues such as the biological characteristics, replication mechanism, genetic characteristics, and immunological characteristics of PDCoV. There is no efficient prevention and control technology, let alone the research of effective drugs and efficient vaccines.

[0003] PDCoV belongs to the order Nidovirales, subfamily Coronavirinae, genus Deltacoronavirus. It is an enveloped, single-stranded, positive-sense RNA virus with a full-length genome of approximately 25.4 kb, consisting of a 5' cap structure and noncoding region, a 3' noncoding region and a poly(A) tail, and seven open reading frames (ORFs), including ORF1a, ORF1b, S, NS6, E, M, and N genes. ORF1a and ORF1b at the 5' end encode two polyproteins (pp1a and pp1ab), while the five ORFs at the 3' end encode, in order, the spike protein (S), NS6 protein, small envelope glycoprotein (E), membrane glycoprotein (M), and nucleocapsid protein (N). Although the successful construction of infectious clones of various plant viruses or viroids has been reported both domestically and internationally, the construction of full-length infectious cDNA clones is technically demanding and remains challenging. Summary of the Invention

[0004] The present invention aims to provide a PDCoV infectious cDNA clone and a construction method and application. The construction method of the PDCoV infectious cDNA clone is rapid, efficient and stable.

[0005] The present invention provides a method for constructing a PDCoV infectious cDNA clone, comprising the following steps:

[0006] (1) Using PDCoV cDNA as a template, primer pairs were used to amplify several fragments of the PDCoV genome, and the two adjacent fragments amplified contained the same terminal homology arms;

[0007] (2) mixing the linearized eukaryotic expression vector with the plurality of fragments amplified in step (1) and transforming the mixture into yeast cells to screen for positive clones; the two ends of the linearized eukaryotic expression vector respectively contain terminal homology arms identical to the first and last fragments amplified in step (1);

[0008] (3) The positive clones screened in step (2) are electroporated into Escherichia coli to obtain the recombinant plasmid, which is the PDCoV infectious cDNA clone.

[0009] Preferably, step (1) comprises amplifying five fragments of the PDCoV genome using five pairs of primers consisting of PDCoV-F1 and PDCoV-R1, PDCoV-F2 and PDCoV-R2, PDCoV-F3 and PDCoV-R3, PDCoV-F4 and PDCoV-R4, and PDCoV-F5 and PDCoV-R5;

[0010] The nucleotide sequences of PDCoV-F1 and PDCoV-R1, PDCoV-F2 and PDCoV-R2, PDCoV-F3 and PDCoV-R3, PDCoV-F4 and PDCoV-R4, and PDCoV-F5 and PDCoV-R5 are shown in SEQ ID No. 1 to 10, respectively.

[0011] Preferably, the method for preparing the linearized eukaryotic expression vector in step (2) comprises using pYES1L-vector as a template and amplifying pYES1L-F and pYES1L-R using primers to obtain the linearized eukaryotic expression vector;

[0012] The nucleotide sequences of the primer pair pYES1L-F and pYES1L-R are shown in SEQ ID No. 11 to SEQ ID No. 12, respectively.

[0013] The present invention also provides a PDCoV infectious cDNA clone constructed using the above construction method.

[0014] The present invention also provides a method for constructing a recombinant plasmid, comprising the following steps: (1) cutting the flanking sequences of NS6 of the above-mentioned PDCoV infectious cDNA clone based on CRISPR / Cas9 gene editing technology to obtain a linear backbone of a PDCoV infectious cDNA cloning vector;

[0015] (2) Using the above-mentioned PDCoV infectious cDNA clone as a template, PCR amplification was performed using primers MF and MR, as well as HiBiT-F and PDCoV-sgRNA1-R, to obtain M and HiBiT-NS6 gene fragments; wherein the nucleotide sequences of MF and MR, as well as HiBiT-F and PDCoV-sgRNA1-R are shown in SEQ ID No. 13 to SEQ ID No. 16, respectively;

[0016] (3) mixing the linear backbone of the PDCoV infectious cDNA cloning vector described in step (1) with the M and HiBiT-NS6 gene fragments described in step (2), and obtaining a recombinant plasmid after homologous recombination;

[0017] There is no temporal relationship between step (1) and step (2).

[0018] Preferably, the sgRNA used in the CRISPR / Cas9 gene editing technology in step (1) includes PDCoV-sgRNA1 and PDCoV-sgRNA2, and the nucleotide sequences are shown in SEQ ID No. 17 and SEQ ID No. 18, respectively.

[0019] The present invention also provides a recombinant plasmid constructed by the above construction method.

[0020] The present invention also provides infectious PDCoV constructed and rescued using the above-mentioned PDCoV infectious cDNA clone or the above-mentioned recombinant plasmid.

[0021] The present invention also provides a method for rescuing the above-mentioned infectious PDCoV, comprising the following steps: transfecting the above-mentioned PDCoV infectious cDNA clone or the above-mentioned recombinant plasmid into mammalian cells, harvesting the culture, and obtaining rescued infectious PDCoV.

[0022] The present invention also provides infectious PDCoV rescued by the above-mentioned rescue method.

[0023] Beneficial effects: The present invention provides a method for constructing a PDCoV infectious cDNA clone, which divides the PDCoV genome into several fragments with the same terminal homologous arms at the front and back, and completes the cloning construction based on a one-step method of yeast homologous recombination, solving the problem of low efficiency of traditional enzyme cutting and ligation, greatly improving the efficiency, and having the characteristics of high efficiency, rapidity, and stability.

[0024] The infectious cDNA clone of the present invention has good stability and can be directly used to transform the PDCoV genome at the genetic level using CRISPR / Cas9 gene editing, thereby improving the efficiency of constructing recombinant viruses and establishing a fast and effective technical platform for the PDCoV genome. The PDCoV infectious cDNA clone provided by the present invention is directly transfected into mammalian cells to rescue the recombinant virus. The CMV promoter contained in the infectious cDNA clone of the present invention can initiate viral genomic RNA synthesis in cells transfected with DNA, avoiding the in vitro RNA transcription synthesis process, simplifying the virus rescue operation process, and solving the problem of transcript heterogeneity caused by in vitro transcription. The present invention also provides a method for constructing a PDCoV infectious clone that can express HiBiT tag protein. The constructed PDCoV infectious clone that can express HiBiT tag protein does not knock out any PDCoV auxiliary protein, providing a good technical means for in-depth research on the PDCoV infection mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the construction of the recombinant plasmid pYES1L-PDCoV;

[0026] Figure 2 This is the gel electrophoresis result of RT-PCR amplification of PDCoV genome fragment and vector;

[0027] Figure 3 This is the result of restriction enzyme digestion and identification of genetic markers of PDCoV infectious cDNA clone;

[0028] Figure 4 Schematic diagram of the construction of the recombinant plasmid pYES1L-PDCoV-HiBiT-NS6;

[0029] Figure 5 Figure 2 is the sequencing result of the recombinant plasmid pYES1L-PDCoV-HiBiT-NS6;

[0030] Figure 6 This is the result of indirect immunofluorescence detection of nucleocapsid protein expression;

[0031] Figure 7 This is the result of fluorescence signal analysis of recombinant virus cell lysate;

[0032] Figure 8 To show the growth kinetics curves of the rescued virus and the parental virus;

[0033] Figure 9 Figure 2 is the result of plaque assay of rescued virus and parental virus;

[0034] Figure 10 This is the result of identifying the genetic stability of the recombinant virus. DETAILED DESCRIPTION

[0035] The present invention provides a method for constructing a PDCoV infectious cDNA clone, comprising the following steps:

[0036] (1) Using PDCoV cDNA as a template, primer pairs were used to amplify several fragments of the PDCoV genome, and the two adjacent fragments amplified contained the same terminal homology arms;

[0037] (2) mixing the linearized eukaryotic expression vector with the plurality of fragments amplified in step (1) and transforming the mixture into yeast cells to screen for positive clones; the two ends of the linearized eukaryotic expression vector respectively contain terminal homology arms identical to the first and last fragments amplified in step (1);

[0038] (3) The positive clones screened in step (2) are transformed into Escherichia coli, and the obtained recombinant plasmid is the PDCoV infectious cDNA clone.

[0039] The present invention preferably uses PDCoV cDNA as a template and the sequence shown in Table 1 as a primer to divide the PDCoV biological genome into 5 fragments for amplification. In the embodiment of the present invention, the PDCoV CH-HA3-2017 strain is used as an example for illustration. The Genbank accession number of the strain sequence is: MK040455.1. Snapgene software is used to design 5 pairs of specific primers as shown in Table 1, and RT-PCR amplifies F1, F2, F3, F4 and F5 five fragments, wherein fragments F1 and F2, fragment F2 and F3, fragment F3 and F4, and fragment F4 and F5 respectively have terminal homology arms, which can be connected based on homologous recombination.

[0040] Table 1 Primer sequences used in the present invention

[0041]

[0042]

[0043] The eukaryotic vector of the present invention is preferably pYES1L-Vector, and the nucleotide sequence of the pYES1L-Vector is preferably as shown in SEQ ID No. 19. In the embodiment, pYES1L-Vector is used as a template, and primers pYES1L-F and pYES1L-R are used for PCR amplification to obtain a linearized pYES1L vector. The linearized pYES1L vector and fragment F1, fragment F5, and linearized pYES1L vector of the present invention respectively have terminal homology arms. Therefore, in the host cell yeast, a recombinant plasmid carrying the full-length cDNA of PDCoV is assembled through the homologous recombination mechanism. The pYES1L-Vector vector of the present invention contains yeast artificial chromosome and bacterial artificial chromosome elements, has good replication stability in yeast and bacteria, and solves the problem that some coronavirus replicase genes cannot be stably preserved in Escherichia coli. At the same time, a cytomegalovirus (CMV) eukaryotic promoter sequence is inserted upstream of the vector, and a hepatitis delta virus ribozyme (HDV ribozyme) sequence and a bovine growth hormone polyadenylation signal (BGH) transcription termination sequence are inserted downstream, greatly improving the efficiency of virus rescue.

[0044] The present invention preferably mixes the linearized pYES1L vector and the five genomic fragments obtained in step (1) and co-transforms the yeast competent cells. After the transformation, the cells are preferably inoculated on a tryptophan (Trp)-deficient plate, and positive clones are screened by colony PCR. The yeast lysate of the positive clone is transformed into a TOP10 electroporation competent cell to obtain a recombinant plasmid, i.e., a PDCoV infectious cDNA clone.

[0045] The present invention also provides a PDCoV infectious cDNA clone constructed using the above construction method.

[0046] In the embodiment of the present invention, after obtaining the above-mentioned recombinant plasmid, it is preferred to further include screening positive recombinants by Spectinomycin resistance, and purifying the recombinant plasmid carrying the full-length cDNA of PDCoV by a plasmid midi extraction kit after proliferation, and naming it pYES1L-PDCoV; and when verifying, a genomic fragment containing a genetic marker is amplified by RT-PCR, and the PCR product is digested and identified with the restriction endonuclease ClaI, and the digestion result is identified by 0.8% agarose gel electrophoresis. Based on the prediction of Snapgene software, the digestion result should contain two fragments of 582 bp and 276 bp respectively.

[0047] The present invention also provides a method for constructing a recombinant plasmid, comprising the following steps: (1) cutting the flanking sequences of NS6 of the above-mentioned PDCoV infectious cDNA clone based on CRISPR / Cas9 gene editing technology to obtain a linear backbone of a PDCoV infectious cDNA cloning vector;

[0048] (2) Using the above-mentioned PDCoV infectious cDNA clone as a template, PCR amplification was performed using primers MF and MR, as well as HiBiT-F and PDCoV-sgRNA1-R, to obtain M and HiBiT-NS6 gene fragments; wherein the nucleotide sequences of MF and MR, as well as HiBiT-F and PDCoV-sgRNA1-R are shown in SEQ ID No. 13 to SEQ ID No. 16, respectively;

[0049] (3) mixing the linear backbone of the PDCoV infectious cDNA cloning vector described in step (1) with the M and HiBiT-NS6 gene fragments described in step (2), and obtaining a recombinant plasmid after homologous recombination;

[0050] There is no temporal relationship between step (1) and step (2).

[0051] The present invention preferably designs two guide RNAs, PDCoV-sgRNA1 and PDCoV-sgRNA2, based on CRISPR / Cas9 gene editing technology to cut the sequences on both sides of NS6 in pYES1L-PDCoV to obtain a linear backbone of a PDCoV infectious cDNA cloning vector.

[0052] The present invention preferably uses pYES1L-PDCoV as a template and uses primers MF / MR and PDCoV-HiBiT-F / PDCoV-sgRNA1-R to perform PCR amplification to obtain M and HiBiT-NS6 gene fragments.

[0053] The present invention preferably uses Cas9 Nuclease and PDCoV-sgRNA 1 and PDCoV-sgRNA2 to cut the pYES1L-PDCoV plasmid to obtain the PDCoV infectious cDNA clone backbone; then the HiBiT-NS6 gene fragment and the M fragment are mixed with the PDCoV infectious cDNA clone backbone fragment, subjected to in vitro homologous recombination, and transformed into TOP10 electroporation competent cells to obtain the recombinant plasmid pYES1L-PDCoV-HiBiT-NS6.

[0054] The present invention also provides a recombinant plasmid constructed by the above construction method.

[0055] The present invention also provides infectious PDCoV constructed and rescued using the above-mentioned PDCoV infectious cDNA clone or the above-mentioned recombinant plasmid.

[0056] The infectious cDNA clone provided by the present invention can be directly transfected into mammalian cells to rescue recombinant viruses. The CMV promoter contained in the infectious cDNA clone of the present invention can initiate viral genomic RNA synthesis in DNA-transfected cells, avoiding the in vitro RNA transcription synthesis process, simplifying the virus rescue process, and also solving the problem of transcript heterogeneity caused by in vitro transcription.

[0057] The present invention also provides a method for rescuing the above-mentioned infectious PDCoV, comprising the following steps: transfecting the above-mentioned PDCoV infectious cDNA clone or the above-mentioned recombinant plasmid into mammalian cells, harvesting the culture, and obtaining rescued infectious PDCoV.

[0058] In the embodiment of the present invention, the pig kidney cell LLC-PK1 cell line is preferably used to rescue the recombinant virus. In the embodiment, the LLC-PK1 cells are preferably cultured to a cell density of about 80% for transformation, such as mixing the infectious cDNA clone plasmid (recombinant plasmid pYES1L-PDCoV or pYES1L-PDCoV-HiBiT-NS6) and the helper plasmid pCAGGS-PDCoV-N to transform the cells, and after 24 hours of transfection, the cell culture medium is replaced with DMEM medium containing 10 μg / mL trypsin. When typical cytopathic effects appear, the cell supernatant is collected to obtain the rescued virus rPDCoV or rPDCoV-HiBiT-NS6. Both viruses rescued by the present invention can be continuously passaged in LLC-PK1 cells.

[0059] When the present invention performs the above-mentioned transformation, it is necessary to complete it under the action of an auxiliary plasmid. The construction method of the auxiliary plasmid pCAGGS-PDCoV-N preferably includes cloning the coding gene of the PDCoVN protein into the pCAGGS vector to obtain the pCAGGS-PDCoV-N auxiliary plasmid. The coding gene of the PDCoV N protein of the present invention is preferably obtained by PCR amplification, using pYES1L-PDCoV as a template and PDCoV-NF and PDCoV-NR in Table 1 as primer pairs for amplification, and then the obtained N gene fragment is inserted between the SacI and XhoI sites of the pCAGGS vector by conventional homologous recombination, and the ligation product is then transformed into TOP10 Escherichia coli competent cells, and the correct recombinant plasmid pCAGGS-PDCoV-N is screened out by sequencing identification.

[0060] The present invention also provides infectious PDCoV rescued by the above-mentioned rescue method.

[0061] The infectious PDCoV described in the present invention can express nanoluciferase, and HiBiT is used as the tag protein during expression. In addition, the infectious PDCoV described in the present invention does not knock out any PDCoV auxiliary protein during construction and rescue, providing a good technical means for in-depth research on the PDCoV infection mechanism.

[0062] To further illustrate the present invention, a PDCoV infectious cDNA clone, construction method and application provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] according to Figure 1 The process shown below is used to construct the infectious cDNA clone plasmid of PDCoV CH-HA3-2017 strain

[0065] According to the full-length genome sequence of PDCoV CH-HA3-2017 strain (Genbank accession number: MK040455.1), 5 pairs of specific primers (Table 1) were designed using Snapgene software to RT-PCR amplify the five fragments F1, F2, F3, F4 and F5, respectively.

[0066] Viral genomic RNA from the PDCoV CH-HA3-2017 strain was extracted according to the instructions of the Viral Genomic DNA / RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). Using viral genomic RNA as a template, PDCoV CH-HA3-2017 cDNA was prepared according to the SuperScript™ IV First-Strand cDNA Synthesis Reaction (Thermo Fisher Scientific). The reaction system consisted of 1 μL of 2 μM gene-specific reverse primer (PDCoV-R7), 1 μL of 10 μM dNTPMix, and 11 μL of template RNA. The reaction procedure was: 65°C for 5 min, followed by 1 min on ice. The reaction system then consisted of 4 μL of 5× SSIV Buffer, 1 μL of 100 mM DTT, 1 μL of Recombinant RNase Inhibitor, and 1 μL of Reverse Transcriptase (200 U / μL). The reaction procedure was: 50°C for 10 min, 80°C for 10 min, and then cooled to room temperature. RNA was removed by adding 1 μL of RNase H and incubating at 37°C for 20 min.

[0067] Using PDCoV cDNA as a template, primers PDCoV-F1 / PDCoV-R1, PDCoV-F2 / PDCoV-R2, PDCoV-F3 / PDCoV-R3, PDCoV-F4 / PDCoV-R4, and PDCoV-F5 / PDCoV-R5 were used for amplification. The reaction system was 50 μL: 1 μL cDNA, 0.625 μL each of upstream primer (40 μM) and downstream primer (40 μM), 1 μL 10 mM dNTPMix, 10 μL 5×Q5 Reaction Buffer, 0.5 μL Q5 High Fidelity DNA Polymerase, and ddH2O to 50 μL. The amplification program was as follows: 98°C for 30 seconds; 98°C for 10 seconds, 62°C-64°C for 20 seconds, 72°C for 5 minutes, 30 cycles; 72°C for 2 minutes. The target band is as follows: Figure 2 As shown, the sizes are 5100 bp, 5090 bp, 5051 bp, 5438 bp and 4949 b, respectively.

[0068] Using pYES1L-Vector as a template, primers pYES1L-F and pYES1L-R were used for PCR amplification to obtain the linearized pYES1L vector. The reaction system is: pYES1L-vector 10ng, upstream primer (40μM) and downstream primer (40μM) 0.625μL each, 10mM dNTPMix 1μL, 5×Q5 Rection Buffer 10μL, Q5 High Fidelity DNA Polymerase 0.5μL, ddH2O to 50μL. The amplification program is: 98℃30s; 98℃10s, 63℃20s, 72℃5min, 30 cycles; 72℃2min. The target band is as follows Figure 2 As shown, the size should be 10257 bp.

[0069] The linearized pYES1L vector was mixed with the F1-F5 fragment and transformed into yeast by the lithium acetate transformation method. The specific operation is as follows: take 100 ng of linearized vector pYES1L and mix it evenly with the above fragments F1, F2, F3, F4, and F5; then take out MaV203 yeast competent cells from -80℃ and thaw them at 30℃ for no more than 90s, add the above mixture to MaV203 yeast competent cells, flick the tube wall to mix it, then add 600 μL PEG / LiAc to the mixture of DNA and yeast competent cells, gently invert it to mix it, and then place it in a 30℃ water bath to incubate for 30 minutes, and gently invert it every 10 minutes to resuspend the mixture; after the incubation, add 35.5 μL DMSO, gently invert it to mix it, and then place it in a 42℃ water bath for heat shock for 20 minutes, and gently invert it every 5 minutes to mix it; after the heat shock, centrifuge it at 1800rpm (200-400g) for 5 minutes, carefully discard the supernatant, and use 1 mL Resuspend the yeast cells in 0.9% NaCl solution; apply 100 μL to a tryptophan-deficient yeast culture dish and culture at 30°C for 2-3 days.

[0070] A single colony was picked and placed in a PCR tube containing 15 μL of Lysis Buffer. The yeast was lysed by gently pipetting three times. 5 μL was transferred to a new PCR tube and stored at 4°C. The remaining 10 μL was used for colony PCR. Positive clones were screened and then electroporated into a TOP10 electrocompetent medium. Positive recombinants were selected for Spectinomycin resistance. After proliferation, the recombinant plasmid carrying the full-length PDCoV cDNA was purified using a plasmid midi extraction kit and named pYES1L-PDCoV.

[0071] The genomic fragment containing the genetic marker was amplified by RT-PCR, and the PCR product was digested with the restriction endonuclease ClaI and the digestion results were confirmed by 0.8% agarose gel electrophoresis. Based on the prediction of Snapgene software, the digestion result should contain two fragments of 582 bp and 276 bp respectively. The results are as follows Figure 3 shown.

[0072] Example 2

[0073] based on Figure 4 The process shown in the figure uses CRISPR / Cas9 gene editing technology to construct the recombinant plasmid pYES1L-PDCoV-HiBiT-NS6

[0074] Based on CRISPR / Cas9 gene editing technology, we designed and commissioned Nanjing GenScript Biotechnology Co., Ltd. to synthesize the two guide RNAs PDCoV-sgRNA1 and PDCoV-sgRNA2 listed in Table 1. These guide RNAs were used to cut the sequences on both sides of NS6 in pYES1L-PDCoV to obtain a linear backbone of the PDCoV infectious cDNA cloning vector.

[0075] The specific operation is: mix 5μL of Cas9 Nuclease Buffer, 3μL each of PDCoV-sgRNA 1 (300nM) and PDCoV-sgRNA 2 (300nM), and 3μL of Cas9 Nuclease, add 3μg of recombinant plasmid pYES1L-PDCoV after 10min at 37℃, and after 2h at 37℃, recover the linearized PDCoV infectious cDNA cloning vector backbone.

[0076] Using pYES1L-PDCoV as a template, PCR amplification was performed with primers MF and MR, HiBiT-F, and PDCoV-sgRNA1-R in Table 1 to obtain the M and HiBiT-NS6 gene fragments.

[0077] The purified vector backbone, M and HiBiT-NS6 fragments were recovered and assembled using NEB's HiFi DNA Assembly Master Mix by in vitro homologous recombination (refer to the kit instructions). The recombinant plasmid was electroporated into TOP10 electroporated competent cells, and positive clones were screened by colony PCR and sequenced. The results are as follows: Figure 5 shown.

[0078] Example 3

[0079] Construction of helper plasmids

[0080] Targeting the open reading frame region of the PDCoV N gene, primers PDCoV-NF and PDCoV-NR (see Table 1) were designed using Snapgene software. PCR amplification was performed using pYES1L-PDCoV as a template to obtain the N gene fragment. The recovered and purified N gene fragment was inserted into the pCAGGS vector between the SacI and XhoI sites via conventional homologous recombination. The ligation product was then transformed into TOP10 competent Escherichia coli cells, and the correct recombinant plasmid, pCAGGS-PDCoV-N, was identified by sequencing. Plasmids were extracted according to the instructions of an endotoxin-free plasmid extraction kit, the concentration was determined, and aliquots were stored at -20°C.

[0081] Example 4

[0082] Rescue and biological characterization of PDCoV CH-HA3-2017 recombinant virus and HiBiT-tagged virus

[0083] 1. Rescue and Passaging of Recombinant Viruses

[0084] LLC-PK1 cells were seeded in 12-well cell culture plates (DMEM supplemented with 10% fetal bovine serum). Once the cell density reached approximately 80%, plasmid transfection was performed according to the instructions for Lipofectamine 3000 transfection reagent. The following plasmid dosages per well were used: 2 μg of the infectious cDNA clone plasmid (recombinant plasmid pYES1L-PDCoV or pYES1L-PDCoV-HiBiT-NS6) and 0.5 μg of the helper plasmid pCAGGS-PDCoV-N. Twenty-four hours after transfection, the cell culture medium was replaced with DMEM supplemented with 10 μg / mL trypsin.

[0085] When typical cytopathic effects appeared, the cell supernatant was collected to obtain the rescued viruses rPDCoV and rPDCoV-HiBiT-NS6. The rescued viruses were serially passaged in LLC-PK1 cells, and each generation of culture was stored at -80°C.

[0086] 2. Identification of Recombinant Viruses

[0087] 2.1 Indirect immunofluorescence detection of viral N protein expression

[0088] The cells after collecting the viral supernatant were fixed with frozen methanol at -20°C for 20 minutes. After the cells were completely dried, monoclonal antibodies against PDCoVN protein (purchased from Medgene Labs) were added, incubated at 37°C for 1 hour, washed 5 times with PBS, and then Goat Anti-Mouse IgG (H+L) Fluor594-conjugated secondary antibodies (purchased from Affinity Biosciences) were added. The cells were incubated at 37°C for 1 hour, washed 5 times with PBS, stained with DAPI for 5 minutes, washed twice with PBS, and observed and recorded the experimental results under a fluorescence microscope. The results are shown in Figure 2. Figure 6 As shown, specific red fluorescence was observed by indirect immunofluorescence using an antibody against the PDCoV N protein, indicating that the recombinant virus rPDCoV was successfully rescued.

[0089] 2.2 RT-PCR identification

[0090] Recombinant virus (rescued virus rPDCoV-HiBiT-NS6) RNA was extracted and amplified using a one-step RT-PCR kit (Nanjing Novozyme Biotechnology Co., Ltd.) using primers MF and PDCoV-SEQ-R10. The fragments were recovered after 1% agarose gel electrophoresis and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing to compare whether the sequence was consistent with the design. The results are as follows Figure 5 As shown, the amplified sequence was consistent with the viral cDNA plasmid sequence.

[0091] 2.3 Fluorescence signal analysis of cell lysate

[0092] LLC-PK1 cells were seeded into 12-well plates and cultured in a 37°C incubator containing 5% CO2. Infection was performed when the cell density reached 100%. LLC-PK1 cells were infected with rPDCoV P3 and rPDCoV-HiBiT-NS6 P3 at an MOI of 0.01. Cell lysates were collected 36 hours later. LgBiT protein was diluted with 1:100 and nanoparticles. HiBiT cleavage substrate was diluted 1:50 in 150 μL of nanoparticles. Place the mixture in HiBiT lysis buffer in a new tube and invert to mix. Add rPDCoV, rPDCoV-HiBiT-NS6, and Mock to a 96-well tube with a white bottom, with three replicates for each sample. Add the mixed reaction solution to the sample at a ratio of 1:1, wait for 10 minutes at room temperature, and then use a microplate reader to detect luciferase activity. The results are as follows. Figure 7 As shown, NanoBiT luciferase activity can be clearly detected in the cell lysate.

[0093] 3. Identification of Virus Biological Characteristics

[0094] 3.1 Virus growth kinetics curve

[0095] LLC-PK1 cells were seeded into 24-well plates and cultured in a 37°C incubator with 5% CO₂. Infection was performed when the cell density reached 100%. PDCoV CH-HA3-2017 P5, rPDCoV P3, and rPDCoV-HiBiT-NS6 P3 were infected with LLC-PK1 cells at an MOI of 0.01, with ten wells infected with each virus. Viral supernatants were collected from two wells at 12, 24, 36, 48, and 60 hours post-infection and stored at -80°C until further use. The collected viral supernatants were serially diluted 10-fold. The infected LLC-PK1 cells were washed twice with PBS to remove serum from the culture medium. The diluted virus solution was then added to 96-well plates, with 100 μL added to each well. Four replicates were set up for each dilution. After a 2-hour incubation, the culture medium was discarded, and 100 μL of DMEM medium containing 10 μg / mL trypsin was added to each well and cultured at 37°C. Observe and record the lesions daily. Observe the cytopathic effect 7 days after infection and calculate the TCID according to the Reed-Muench method. 50 , draw the growth curve of the virus.

[0096] Growth curves such as Figure 8 As shown in the figure, compared with the parental virus, the growth characteristics of the recombinant viruses (rPDCoV and rPDCoV-HiBiT-NS6) on cells are basically the same. Further, the cell lysates at different time points after the recombinant virus rPDCoV-HiBiT-NS6 infected cells were used to measure the NanoBiT luciferase activity. The results showed that at different time points after infection, as the titer of the recombinant virus rPDCoV-HiBiT-NS6 increased, the NanoBiT luciferase activity value also increased and reached a peak at 36h, with titers of 10 7.7 and 10 7.1 .

[0097] 3.2 Viral plaque-forming ability

[0098] ST cells were seeded into 12-well plates and cultured in a 37°C incubator containing 5% CO2. Infection was performed when the cell density reached 100%. PDCoV CH-HA3-2017 P5, rPDCoV P3, and rPDCoV-HiBiT-NS6 P3 were diluted 10-fold with DMEM medium, and the cells to be infected were washed twice with PBS to remove the serum in the culture medium. The diluted virus solution was added to a 12-well plate and incubated at 37°C and 5% CO2. After 2 hours of infection, the virus solution was discarded, and 2×DMEM (containing 20 μg / mL trypsin) and 2% UltraPureTM LMPAgarose were mixed in equal proportions and added to the 12-well plate. After complete solidification, the plate was inverted and cultured in a 37°C, 5% CO2 incubator. Three days after infection, fix the cells with 4% paraformaldehyde overnight. The next day, discard the 4% paraformaldehyde and gel, add 300 μL of 1% crystal violet staining solution to each well, stain for 10 minutes, discard the crystal violet solution, wash with water, and take photos after drying.

[0099] The results are as follows Figure 9 As shown, no significant differences in size and morphology were observed among the plaques formed by the three strains.

[0100] 3.3 Viral genetic stability

[0101] To verify the genetic stability of the HiBiT tag in the recombinant virus, rPEDV-HiBiTNS6 was serially passaged in LLC-PK1 cells, and the genetic stability of the P3 and P10 viruses was tested. As shown in the figure, the HiBiT gene of the P3 and P10 viruses was amplified by RT-PCR and verified by DNA sequencing. The results showed that the HiBiT sequence was stably present in the P3 and P10 viruses, indicating that the recombinant virus is genetically stable in vitro.

[0102] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for constructing an infectious cDNA clone of swine delta coronavirus, characterized in that: The following steps are involved: (1) Using the cDNA of swine delta coronavirus as a template, five fragments of the swine delta coronavirus genome are amplified using a primer pair, and the two adjacent fragments amplified contain the same terminal homology arms; the primer pair includes PDCoV-F1 and PDCoV-R1, PDCoV-F2 and PDCoV-R2, PDCoV-F3 and PDCoV-R3, PDCoV-F4 and PDCoV-R4, and PDCoV-F5 and PDCoV-R5; wherein the nucleotide sequences of PDCoV-F1 and PDCoV-R1, PDCoV-F2 and PDCoV-R2, PDCoV-F3 and PDCoV-R3, PDCoV-F4 and PDCoV-R4, and PDCoV-F5 and PDCoV-R5 are shown in SEQ ID No. 1 to 10 respectively; (2) mixing the linearized eukaryotic expression vector with the plurality of fragments amplified in step (1), transforming the mixture into yeast cells, and screening positive clones; the two ends of the linearized eukaryotic expression vector respectively contain terminal homology arms identical to the first and last fragments amplified in step (1); the method for preparing the linearized eukaryotic expression vector comprises using pYES1L-vector as a template and using primer pairs pYES1L-F and pYES1L-R to amplify the linearized eukaryotic expression vector; wherein the nucleotide sequences of the primer pairs pYES1L-F and pYES1L-R are shown in SEQ ID No.11 to SEQ ID No.12, respectively; (3) The positive clones screened in step (2) are transformed into Escherichia coli, and the obtained recombinant plasmid is the infectious cDNA clone of the swine delta coronavirus.

2. A method for constructing a recombinant plasmid, characterized in that: The method comprises the following steps: (1) cutting the sequences on both sides of NS6 of the infectious cDNA clone of swine delta coronavirus based on CRISPR / Cas9 gene editing technology to obtain a linear backbone of a cloning vector lacking the infectious cDNA clone of swine delta coronavirus; the infectious cDNA clone of swine delta coronavirus is constructed according to the construction method of claim 1; (2) Using the swine delta coronavirus infectious cDNA clone as a template, PCR amplification was performed using primer pairs MF and MR, as well as HiBiT-F and PDCoV-sgRNA1-R, to obtain M and HiBiT-NS6 gene fragments; wherein the nucleotide sequences of MF and MR, as well as HiBiT-F and PDCoV-sgRNA1-R are shown in SEQ ID No. 13 to SEQ ID No. 16, respectively; (3) mixing the linear backbone of the cDNA cloning vector lacking the infectious cDNA of swine delta coronavirus described in step (1) with the M and HiBiT-NS6 gene fragments described in step (2), and obtaining a recombinant plasmid after homologous recombination; There is no temporal relationship between step (1) and step (2).

3. The construction method according to claim 2, characterized in that: The sgRNA used in the CRISPR / Cas9 gene editing technology described in step (1) includes PDCoV-sgRNA1 and PDCoV-sgRNA2, and the nucleotide sequences are shown in SEQ ID No.17 and SEQ ID No.18 respectively.

4. An infectious swine delta coronavirus cDNA clone constructed using the construction method of claim 1 or an infectious swine delta coronavirus constructed and rescued using a recombinant plasmid constructed using the construction method of claim 2 or 3.

5. The method for rescuing infectious swine delta coronavirus according to claim 4, characterized in that: The method comprises the following steps: transfecting mammalian cells with the infectious cDNA clone of swine delta coronavirus constructed by the construction method described in claim 1 or the recombinant plasmid constructed by the construction method described in claim 2 or 3, harvesting the culture, and obtaining rescued infectious swine delta coronavirus.

Images