Method for rapid rescue of bovine coronavirus epidemic strain and application thereof

Abstract

This invention discloses a method for rapidly rescuing prevalent bovine coronavirus strains and its applications. The invention involves efficient fragment cloning of the bovine coronavirus SHZ isolate, followed by circular polymerase extension of the resulting fragments with a linker sequence. The resulting product can be directly transfected into 293T cells for viral packaging without purification. Unlike traditional methods, this invention eliminates the need for bacteria and yeast to amplify the full-length viral cDNA clone. Instead, it directly obtains a sufficient quantity of preliminary product via PCR, avoiding the instability and inefficiency associated with partial viral genome replication in bacterial or yeast hosts. The successful rescue of recombinant bovine coronavirus expressing exogenous proteins using this reverse genetics system provides a novel technical platform for visualizing viral replication dynamics both in vivo and in vitro, and offers an efficient and flexible tool for virological research, viral vector vaccine development, and antiviral drug screening.

Description

Technical Field

[0001] This invention belongs to the fields of virology and molecular biology, specifically relating to a method for rapidly rescuing prevalent bovine coronavirus strains, its products, and applications. Background Technology

[0002] Bovine Coronavirus (BCoV) is a lung-enteric pathogen of cattle that is widely distributed globally. It causes winter dysentery and calf diarrhea in cattle and is associated with respiratory diseases in cattle.

[0003] Bovine coronavirus (BCoV) belongs to the family Coronaviridae, genus Betacoronavirus, and is closely related to human coronavirus OC43. BCoV is a single-stranded positive-sense RNA virus with a genome larger than 30 kb. Approximately two-thirds of the 5' end of the genome encodes a large polypeptide protein (pp), while the remaining third primarily encodes five structural proteins, including hemagglutinin esterase protein (HE), spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N), as well as several accessory proteins (ns2, ns4.9, ns4.8, ns12.7). Similar to other coronaviruses, due to its high mutation rate and recombination frequency, BCoV can rapidly adapt to its host and has been reported to infect giraffes, sheep, and other ruminants. Therefore, BCoV has attracted increasing attention due to its wide host range and the economic losses it causes.

[0004] Reverse genetics is an important technique for studying viral genome function and viral infection mechanisms. It primarily involves using molecular biology techniques to reverse-engineer gene sequences from the viral genome to reconstruct the virus, thereby studying gene function or altering viral characteristics. It is also an indispensable tool for vaccine development and antiviral drug development. The ongoing pandemics caused by recurring coronaviruses necessitate a rapid and easily implementable reverse genetics platform capable of reliably assembling viral genomes to quickly rescue viruses and their mutants for characterization studies.

[0005] The systems reported to be successful for cloning full-length cDNA of animal coronaviruses include: (1) in vitro enzyme digestion and ligation; (2) bacterial artificial chromosome (BAC) system; (3) yeast artificial chromosome (YAC) system; and (4) poxvirus vector system. Due to the large size of the coronavirus genome and the toxicity of some replicase genes, the existing coronavirus rescue systems each have their own technical hurdles. Although the in vitro ligation method avoids the cloning of the viral genome in bacteria, the efficiency of large-fragment enzyme digestion and ligation in vitro is low, and the transcripts obtained by in vitro transcription are heterogeneous, resulting in low transfection efficiency. The whole process is cumbersome and time-consuming. Virus rescue based on bacterial artificial chromosomes requires the construction of multiple intermediate plasmids. It is necessary to ensure that the sequence of each intermediate plasmid constructed is accurate in order to finally assemble a complete full-length cDNA clone. The workload is large and the gene sequencing cost is high. Although the low copy number of BAC plasmids greatly avoids the instability of coronaviruses, toxic or unstable sequences are still easily generated during the operation, leading to construction failure. The operation technology of rescuing coronaviruses based on yeast artificial chromosomes (YAC) is much more complicated than that of E. coli. The transformation efficiency is usually low, and the screening of positive clones is more time-consuming and labor-intensive. Furthermore, isolating and purifying complete YAC plasmids (containing large exogenous fragments) is difficult and yields low results. In some cases, large exogenous fragments are prone to partial deletion or rearrangement during yeast passage, which is disastrous for virus rescue. While vaccinia virus vector systems can accommodate the full-length genome of coronaviruses, their homologous recombination efficiency is low, and screening and purification are time-consuming.

[0006] Currently, the construction of easily operable full-length viral cDNA clones, the acquisition of infectious viral RNA, and the improvement of virus rescue efficiency are crucial technical challenges that urgently need to be addressed in the field of bovine coronavirus reverse genetics research. Furthermore, the high clinical infection rate but low isolation rate of bovine coronavirus limits the development of its pathogenic mechanisms, immune mechanisms, novel drugs, and vaccines. The development of an efficient reverse genetics platform will greatly promote research progress in various aspects of bovine coronavirus. Summary of the Invention

[0007] The purpose of this invention is to provide a method for rapidly rescuing prevalent bovine coronavirus strains and its application. This method is a reverse genetics technique for rapidly and efficiently rescuing bovine coronavirus, overcoming the shortcomings of existing methods, such as cumbersome construction processes and low virus rescue efficiency. This invention employs circular polymerase extension reaction (CPER) technology to ligate the linker sequences containing each transcription element group and the bovine coronavirus genome sequence end-to-end in the 5'→3' direction, rapidly constructing a circular clone containing the linker and the full-length cDNA of the bovine coronavirus genome. The resulting CPER product requires no purification before transfection into cells, where the viral genome is transcribed and packaged intracellularly, thereby rapidly rescuing and obtaining bovine coronavirus.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0009] In a first aspect, the present invention claims protection for a method for rapidly rescuing a prevalent strain of bovine coronavirus, the method comprising the following steps:

[0010] (1) The genome of the bovine coronavirus SHZ isolate was cloned in segments to obtain six DNA fragments AF;

[0011] (2) Design a Linker sequence, wherein the Linker sequence comprises, in sequence: a 30-50bp bovine coronavirus 3'UTR sequence, a hepatitis D ribozyme sequence (HDVRz), a BGH poly(A) signal sequence, a CMV promoter sequence and a 30-50bp bovine coronavirus 5'UTR sequence;

[0012] (3) Mix the six DNA fragments AF from step (1) with the linker sequence designed in step (2) and perform a CPER reaction to obtain the CPER reaction product;

[0013] (4) The CPER reaction product obtained in step (3) does not need to be purified in vitro and can be directly transfected into cells for virus packaging;

[0014] (5) The transfected cells were co-cultured with virus-proliferating cells to rescue and obtain recombinant bovine coronavirus.

[0015] Furthermore, the nucleotide sequences of the six DNA fragments AF described in step (1) are shown in SEQ ID NO: 1-6.

[0016] As a technical solution, at least one of the DNA fragments in step (1) may further contain a foreign gene sequence. Further, the foreign gene sequence is inserted upstream of the viral ns2 protein gene. Further, a self-cleaving peptide sequence is present between the foreign gene sequence and the ns2 protein gene. Further, the foreign gene is a reporter gene or an antigen-encoding gene. In a specific embodiment of the present invention, the reporter gene is a gene encoding green fluorescent protein ZsGreen, the sequence of which is shown in SEQ ID NO: 12.

[0017] Furthermore, the CMV promoter sequence mentioned in step (2) is shown in SEQ ID NO: 7, the hepatitis D ribozyme sequence (HDVRz) is shown in SEQ ID NO: 8, the BGH poly(A) signal sequence is shown in SEQ ID NO: 9, the 5'UTR sequence is shown in SEQ ID NO: 10, and the 3'UTR sequence is shown in SEQ ID NO: 11.

[0018] Furthermore, the CPER reaction product described in step (4) is directly transfected into 293T cells for virus packaging; in step (5), the transfected 293T cells are co-cultured with HRT-18G cells.

[0019] Secondly, the present invention seeks protection for recombinant bovine coronaviruses rescued by the above-described method.

[0020] Thirdly, the present invention seeks protection for an infectious clonal construct of bovine coronavirus full-length cDNA, the construct comprising six fragments A to F of the bovine coronavirus genome with nucleotide sequences as shown in SEQ ID NO:1-6 and a linker sequence for linking fragments A and F, wherein the six fragments A to F and the linker sequence are mixed and subjected to a CPER reaction, and the CPER reaction product obtained is the infectious clonal construct of bovine coronavirus full-length cDNA. Furthermore, the linker sequence sequentially comprises: a 3'UTR sequence of 30-50 bp bovine coronavirus, a hepatitis D ribozyme sequence (HDVRz), a BGH poly(A) signal sequence, a CMV promoter sequence, and a 5'UTR sequence of 30-50 bp bovine coronavirus; the CMV promoter sequence is shown in SEQ ID NO: 7, the hepatitis D ribozyme sequence (HDVRz) is shown in SEQ ID NO: 8, the BGH poly(A) signal sequence is shown in SEQ ID NO: 9, the 5'UTR sequence is shown in SEQ ID NO: 10, and the 3'UTR sequence is shown in SEQ ID NO: 11.

[0021] Fourthly, the present invention seeks protection for at least one of the following applications of the above-described recombinant bovine coronavirus: (1)-(3)

[0022] (1) Application in the preparation of viral vector vaccines;

[0023] (2) Use as a reporter virus in the preparation of vaccines or diagnostic reagents for bovine coronavirus prevention and control;

[0024] (3) Application in the screening of anti-bovine coronavirus drugs.

[0025] In a specific embodiment of the present invention, a method for rapidly rescuing prevalent bovine coronavirus strains (e.g.) Figure 1 (As shown) Specifically, it includes the following steps:

[0026] (1) The genome of bovine coronavirus (BCoV) SHZ isolate was rapidly segmented and cloned into fragments A, B, C, D, E and F, and cloned into the pBR322 vector respectively. Fragment E contains the complete viral Spike gene.

[0027] (2) Design and construct a plasmid containing a linker sequence: After the designed linker sequence is synthesized by the company, it is cloned into the pcDNA3.1 vector according to the homologous recombination method. The linker sequence includes the following elements in sequence: 30-50bp bovine coronavirus 3'UTR sequence, hepatitis D ribozyme sequence (HDVRz), BGH poly(A) signal sequence, CMV promoter sequence and 30-50bp bovine coronavirus 5'UTR sequence; the CMV promoter sequence is shown in SEQ ID NO: 7, the hepatitis D ribozyme sequence (HDVRz) is shown in SEQ ID NO: 8, the BGH poly(A) signal sequence is shown in SEQ ID NO: 9, the 5'UTR sequence is shown in SEQ ID NO: 10, and the 3'UTR sequence is shown in SEQ ID NO: 11.

[0028] (4) Mix the fragment from step (1) with the linker sequence designed in step (2) and perform a CPER reaction to obtain the pre-transfected DNA product;

[0029] (5) CPER products do not need to be purified in vitro. They are directly transfected into 293T cells for viral packaging. Then, the transfected 293T cells are co-cultured with HRT-18G cells to amplify progeny viruses. The viruses produced by the co-cultured cells are referred to as P1 generation viruses.

[0030] (6) Continue blind transmission to generation P3 to allow the virus to proliferate stably.

[0031] In a specific embodiment of the present invention, a method for constructing recombinant bovine coronavirus reporter virus rBCoV SHZ-ZsGreen expressing green fluorescent protein ZsGreen is also provided, comprising the following steps:

[0032] (1) Repeat step (1) above, and modify fragment D in step (1) by inserting the green fluorescent reporter gene ZsGreen upstream of the ns2 gene and downstream of the transcriptional regulatory sequence (TRS) of the ns2 gene, and inserting the T2A peptide between ZsGreen and the ns2 gene. This fragment is named D-ZsGreen. The ZsGreen sequence is shown in SEQ ID NO: 12.

[0033] (3) Repeat steps (2) to (6) above to obtain bovine coronavirus recombinant reporter virus expressing reporter gene.

[0034] The method of this invention enables rapid segmented cloning of the bovine coronavirus genome, facilitating subsequent reverse genetics operations. Based on the segmented cloned bovine coronavirus genome, a circular polymerase reaction is performed to rapidly rescue clinical isolates of bovine coronavirus, without relying on instability caused by vectors such as bacteria, yeast, or poxviruses.

[0035] The method of this invention can rescue the bovine coronavirus reverse genetics rescue strain rSHZ; by inserting a foreign gene (such as the ZsGreen fluorescent protein gene) into the viral genome (such as before the ns2 gene) using the method of this invention, a virus expressing fluorescent protein, rBCoV SHZ-ZsGreen, can also be rescued. This makes it possible to directly observe the dynamic process of viral infection under a microscope. It can be applied to research on bovine coronavirus gene expression, vector vaccine preparation, and antiviral drug screening.

[0036] Compared with existing coronavirus reverse genetics technology, the present invention has the following significant advantages:

[0037] (1) Instead of using bacteria and yeast to amplify the full-length viral cDNA clone, sufficient preliminary products can be obtained directly through PCR, avoiding the instability of the viral genome sequence during proliferation in bacterial or yeast hosts and the problem of low in vitro ligation efficiency, which greatly shortens the time for constructing infectious clones; (2) The final infectious clone can be directly introduced into 293T cells without any purification steps, and the virus can be quickly packaged to produce infectious progeny viruses. The recombinant virus obtained can be stably passaged and proliferated; (3) It can simultaneously modify different viral genome sequences, providing an efficient platform for the development of genetic engineering vector vaccines.

[0038] The bovine coronavirus (BCoV) reverse genetics platform construction method provided by this invention can quickly and efficiently rescue clinically circulating strains, providing an efficient and flexible tool for the study of the pathogenic mechanism of bovine coronavirus, the development of attenuated live vaccines, and the screening of antiviral drugs. It solves the technical problems of BCoV in basic and applied research and has important practical application value. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of a method for rescuing bovine coronavirus based on cyclic polymerase reaction (CPER).

[0040] Figure 2 This is a schematic diagram of CPE of the recombinant virus and parent virus in HRT-18G cells in Example 1.

[0041] Figure 3 This is the identification of genetic markers for the recombinant virus in Example 1.

[0042] Figure 4This is the immunofluorescence identification of the recombinant virus and the parent virus in Example 1.

[0043] Figure 5 This is a Western blot identification of the recombinant virus and the parent virus N protein in Example 1.

[0044] Figure 6 This is the titer determination of recombinant virus and parent virus in Example 1.

[0045] Figure 7 This is a graph showing the replication kinetics of the recombinant virus and the parent virus in Example 1.

[0046] Figure 8 This is a schematic diagram of the insertion of the green fluorescent reporter gene ZsGreen into the bovine coronavirus genome in Example 2.

[0047] Figure 9 This is the replication kinetic curve of the virus and its parent virus reported in Example 2.

[0048] Figure 10 Example 2 reports the cellular fluorescence observation of the report virus rBCoV-SHZ-ZsGreen and the parent virus BCoV-SHZ, as well as the passaging stability of the report virus.

[0049] Figure 11 This is Example 2, which describes the stability of nucleic acid detection in the P10 generation reporter gene. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. Modifications or substitutions to the details and form of the present invention may be made without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0051] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0052] The bovine coronavirus epidemic strain SHZ was isolated, preserved, and donated by the Xinjiang Academy of Animal Sciences; human embryonic kidney cells (293T) and human colorectal adenocarcinoma cells (HRT-18G) are preserved in our laboratory's cell bank, and primers and gene sequences were synthesized by General Biotech.

[0053] Example 1: Rapid segmentation cloning and virus rescue of bovine coronavirus genome

[0054] 1. Experimental Methods

[0055] 1.1 Primer Design and Synthesis

[0056] Based on the gene sequence provided by the Genbank accession number (Gene ID: OR750853.1) of the prevalent bovine coronavirus strain SHZ, primers were designed using Oligo 7 software to divide BCoV into six fragments AF (BCoV-A nucleotide sequence as shown in SEQ ID NO: 1, BCoV-B nucleotide sequence as shown in SEQ ID NO: 2, BCoV-C nucleotide sequence as shown in SEQ ID NO: 3, BCoV-D nucleotide sequence as shown in SEQ ID NO: 4, BCoV-E nucleotide sequence as shown in SEQ ID NO: 5, and BCoV-F nucleotide sequence as shown in SEQ ID NO: 6). Each fragment contains a 35-40 bp overlap region. The primer sequences are shown in Table 1. To distinguish the recombinant virus from the parental virus, a silent mutation, T29408C, was introduced into the BCoV-SHZ-F fragment as a genetic marker through site-directed mutagenesis.

[0057] Table 1 Primer Sequences

[0058]

[0059] 1.2 Segmented Cloning of the BCoV Genome

[0060] RNA was extracted from 500 μL of viral culture supernatant. The obtained RNA was then reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, R212) to obtain viral cDNA. The BCoV-SHZ-AF fragments (as shown in SEQ ID NO: 1-6) were amplified by PCR using the primers in Table 1 and Phanta Max high-fidelity DNA polymerase (Vazyme, catalog number: P505). Each of the six fragments was cloned into the pBR322 vector and stored.

[0061] A linker sequence was designed and synthesized, comprising, sequentially, the last 30-50 bp of the BCoV SHZ 3'UTR sequence (as shown in SEQ ID NO: 11), the hepatitis D ribozyme sequence (HDVRz, as shown in SEQ ID NO: 8), the BGH poly(A) signal sequence (as shown in SEQ ID NO: 9), the CMV promoter (as shown in SEQ ID NO: 7), and the first 30-50 bp of the BCoV SHZ 5'UTR sequence (as shown in SEQ ID NO: 10). The linker sequence was cloned into the pcDNA3.1(+) vector using EcoRI and XhoI restriction endonuclease sites and stored, named pcDNA3.1-SHZ-Linker, and validated in full length using Sanger sequencing.

[0062] 1.3 CPER reaction

[0063] The seven fragments generated by PCR were subjected to CPER reaction to produce circular products. Equimolar amounts of the seven fragments (A to F, linker sequences) were reacted with NEB Q5 high-fidelity DNA polymerase in a 50 μL reaction volume for CPER. The specific CPER reaction system was: 10 μL 5X Q5 reaction buffer, 1 μL dNTP Mix, DNA (AF and linker), 2 μL Q5 DNA polymerase, and 50 μL ddH2O. The CPER reaction conditions were as follows: initial denaturation at 98℃ for 3 min; 5 cycles of 98℃ for 15 s, 55℃ for 15 s, and 72℃ for 10 min; followed by 15 cycles of 98℃ for 15 s, 55℃ for 15 s, and 72℃ for 15 min. The CPER products were temporarily stored at 4℃.

[0064] 1.4 CPER Transfection and Virus Rescue

[0065] CPER product can be directly transfected into 293T cells for virus packaging without any purification steps. Transfection can begin when 293T cells reach a confluence of more than 80%. Before transfection, replace the 293T cell culture medium with serum-free medium. Then prepare the transfection system by diluting the DNA and transfection reagent separately with culture medium: Take two 0.5mL EP tubes, labeled A and B respectively, and add 150 μL of opti-MEM to each. Dilute the CPER product in tube A and dilute the PEI in tube B. Mix tubes A and B and incubate at room temperature (25±5℃) to form a DNA-transfection reagent complex. After 15 min, add the complex dropwise to 293T cells, shake the plate to distribute the complex evenly, and incubate in a 5% CO2 incubator.

[0066] 293T cells were co-cultured with HRT-18G cells with a confluence of approximately 50-60%. After cell adhesion, the cells were cultured for another 3-6 days. Once typical CPE was observed, the cells were passaged 1-2 times. The passaged virus was further identified by PCR, WB, IFA, etc., and the viral titer was determined. A one-step growth curve was plotted.

[0067] PCR identification: RNA was extracted from the cell supernatant after two blind passages. cDNA was obtained by RT-PCR and then identified using primers for the N and S genes of BCoV, as shown in Table 2. Positive samples were sequenced to identify their genetic markers.

[0068] Table 2 Identification Primers

[0069]

[0070] Western blot identification: HRT-18G cells were infected with the PCR-positive recombinant virus. Positive virus infection control and negative control were also set up. After culturing for 48 h, the cell culture supernatant was discarded, cell debris was washed away with PBS, and NP40 was added to lyse the cells. After 30 min, the cells were centrifuged at 12000 rpm for 10 min. The supernatant was collected and added to 5X Loading Buffer for SDS-PAGE electrophoresis. After electrophoresis, the protein was transferred to an NC membrane and blocked with 5% (w / v) skim milk at room temperature for 1 h. Then, the cells were incubated overnight at 4 °C with BCoV N protein polyclonal antibody (1:1000). The cells were washed 5 times with PBST and incubated at room temperature with secondary antibody goat anti-rabbit HRP-IgG (1:5000) for 1 h. The cells were washed 5 times with PBST and the bands were imaged using a chemiluminescence analyzer.

[0071] IFA identification: HRT-18G cells were infected with the PCR-positive recombinant virus. Positive virus infection control and negative control were also set up. After culturing for 48 h, the cell culture supernatant was discarded, cell debris was washed away with PBS, and cells were fixed with 4% paraformaldehyde. The cells were washed three times with PBS at room temperature for 15 min, permeabilized with 0.1% (v / v) Triton X-100 at room temperature for 15 min, washed three times with PBS, blocked with 5% (w / v) skim milk at room temperature for 1 h, incubated overnight at 4°C with BCoV N protein polyclonal antibody (1:250), washed three times with PBS, and incubated with secondary antibody goat anti-rabbit FITC-IgG (1:1000) at room temperature for 1 h. The secondary antibody was discarded, and the cells were washed three more times with PBS. The cells were observed and photographed under an inverted fluorescence microscope.

[0072] TCID 50Assay: Collect virus cell culture, centrifuge at 5000 rpm for 10 min at 4℃ to discard cell debris, and use the supernatant for 10-fold serial dilution. Seed the supernatant into HRT-18G cells cultured in 96-well plates, with 8 replicates per dilution. Add 100 μL of diluted virus solution to each well and incubate at 37℃ 5% CO2 for 72 h. After 72 h, remove cells for IFA assay and calculate viral TCID using the Karber method. 50 .

[0073] Viral replication kinetics curve determination: HRT-18G cells with confluent monolayers were infected with wt-BCoV-SHZ and rBCoV-SHZ at a dose of 0.01 mol. Cell supernatant was collected at 12 hpi, 24 hpi, 48 hpi, and 72 hpi, respectively. Viral titer was measured, and viral growth curves were plotted to observe viral replication dynamics.

[0074] 2. Experimental Results

[0075] The results showed that the rescued virus rBCoV-SHZ had similar CPE characteristics to its parent virus WT-BCoV-SHZ—cell fusion to form syncytia, cell shedding to form vacuoles, etc. Figure 2 (As shown); PCR identification showed that the rBCoV-SHZ-S and N genes had positive bands at the same positions as WT-SHZ. Sequencing of rBCoV-SHZ revealed that it possessed a genetic marker (T29408C) not found in its parent virus. Figure 3 ), IFA ( Figure 4 ) and WB ( Figure 5 The identification results showed that rBCoV-SHZ exhibited reactivity consistent with WT-BCoV-SHZ—a specific response to the N protein, i.e., specific green fluorescence in virus-positive cells and a specific band at the 55kD protein size; furthermore, the viral titer was determined... Figure 6 The growth curves of the virus at different time points after infection were measured, and it was found that the rescued virus and the parent virus had similar replication kinetics curves. Figure 7 The above results indicate that the virus was successfully rescued.

[0076] Example 2: Preliminary application of the BCoV reverse genetics system

[0077] Rescue of recombinant bovine coronavirus rBCoV-SHZ-ZsGreen expressing the green fluorescent reporter gene.

[0078] Using the aforementioned technical approach, a green fluorescent protein (GFP) gene was inserted into the bovine coronavirus (BCoV) genome. The first two-thirds of the BCoV genome is a large replicase gene, which can be divided into ORF1a and ORF1b, which translate into two large polyproteins, pp1a and pp1ab, respectively. These are then hydrolyzed by a protease to produce the non-structural protein nsp1-16. Downstream of the replicase gene is ORF2a, encoding the accessory protein ns2, a cytoplasmic protein with a molecular weight of 30 kDa. According to reports, the ns2 protein of MHV is not essential for viral replication in vitro, but it is crucial for the severity of hepatitis induced by MHV in mice, suggesting that ns2 may be a viral virulence gene. Furthermore, ns2 antagonizes the production of type I interferon, which may be related to the viral escape mechanism from the host's immune system. To characterize the role of the ns2 protein in the interaction between bovine coronavirus and the host, a GFP gene (ZsGreen) was inserted before the ns2 gene to visualize viral replication dynamics.

[0079] Fragment D containing the expression of ns2 protein was modified. Specifically, the ZsGreen gene (SEQ ID NO:12) was amplified from the pZsGreen1-C1 plasmid template (purchased from Takara) using primers ZsGreen-F and ZsGreen-R. Simultaneously, primers were designed before the ATG start codon of the ORF2a gene at the insertion site of the ZsGreen gene. Using the pBR322-SHZ-D plasmid constructed in the previous stage of this invention as a template, circular PCR amplification was performed to obtain a linear vector. Homologous recombination of the vector and the fragment yielded the pBR322-SHZ-D-ZsGreen plasmid. Through this construction scheme, ZsGreen can be placed under the regulation of the transcriptional regulatory sequence (TRS-B) of the ORF2a gene expression cassette, allowing the exogenous ZsGreen gene to stably exist in the viral genome. A detailed schematic diagram is shown below. Figure 8 As shown in Table 3. Furthermore, a T2A peptide was inserted between the ZsGreen and ORF2a genes to enable automatic cleavage during translation, achieving independent expression of the two proteins. The primers used are listed in Table 3.

[0080] Table 3 Primer sequences

[0081]

[0082] According to the technical solution of Example 1, the CPER product of SHZ-ZsGreen was prepared.

[0083] The CPER product was transfected into 293T cells using the above-described technique to package the virus. Results showed that the recombinant reporter virus could infect HRT-18G cells and produce CPE identical to the parental virus, and exhibited similar replication kinetics to the parental recombinant virus rBCoV-SHZ. Figure 9 ); Inverted fluorescence microscopy revealed obvious green fluorescence in HRT-18G cells infected with rBCoV-SHZ-ZsGreen, while the recombinant parental virus rSHZ showed no fluorescence ( Figure 10 Even after the virus was passed to generation P10, strong fluorescence could still be observed. Figure 10 ); The ZsGreen gene remained stable in the P10 generation in nucleic acid testing ( Figure 11 ).

[0084] The above results demonstrate that this reverse genetics platform can be used efficiently to rescue recombinant viruses and can stably express exogenous genes. Applying this platform not only provides a new technical platform for the visualization of viral replication dynamics in vivo and in vitro, but also provides an efficient and flexible tool for virological research, the development of viral vector vaccines, and the screening of antiviral drugs.

Claims

1. A method for rapid rescue of bovine coronavirus epidemic strains, characterized in that, The method includes the following steps: (1) The genome of bovine coronavirus SHZ isolate was cloned in segments to obtain six DNA fragments AF; the nucleotide sequences of the six DNA fragments AF are shown in SEQ ID NO: 1-6; (2) Design a linker sequence, wherein the linker sequence comprises, in sequence: a 30-50bp bovine coronavirus 3'UTR sequence, a hepatitis D ribozyme sequence, a BGH poly(A) signal sequence, a CMV promoter sequence and a 30-50bp bovine coronavirus 5'UTR sequence; the CMV promoter sequence is shown in SEQ ID NO: 7, the hepatitis D ribozyme sequence is shown in SEQ ID NO: 8, the BGH poly(A) signal sequence is shown in SEQ ID NO: 9, the 5'UTR sequence is shown in SEQ ID NO: 10, and the 3'UTR sequence is shown in SEQ ID NO: 11; (3) Mix the six DNA fragments AF from step (1) with the linker sequence designed in step (2) and perform a CPER reaction to obtain the CPER reaction product; (4) The CPER reaction product obtained in step (3) does not need to be purified in vitro and can be directly transfected into 293T cells for virus packaging; (5) The transfected 293T cells were co-cultured with HRT-18G cells to rescue and obtain recombinant bovine coronavirus.

2. The method of claim 1, wherein, In step (1), at least one of the DNA fragments contains a foreign gene sequence.

3. The method of claim 2, wherein, The exogenous gene sequence was inserted upstream of the viral ns2 protein gene.

4. The method of claim 3, wherein, The exogenous gene sequence contains a self-cleaving peptide sequence between it and the ns2 protein gene.

5. The method according to claim 2, characterized in that, The exogenous gene is a reporter gene or an antigen-encoding gene.

6. The method according to claim 5, characterized in that, The reporter gene is a gene encoding the green fluorescent protein ZsGreen, the sequence of which is shown in SEQ ID NO:

12.

7. The recombinant bovine coronavirus obtained by the method of any one of claims 1-6.

8. An infectious cDNA construct of bovine coronavirus, characterized in that, The construct includes six fragments A to F of the bovine coronavirus genome with nucleotide sequences as shown in SEQ ID NO:1-6 and a linker sequence for linking fragments A and F. The six fragments A to F and the linker sequence are mixed and subjected to a CPER reaction. The CPER reaction product is the full-length cDNA infectious clone construct of bovine coronavirus. The linker sequence comprises, in sequence: a 3'UTR sequence of 30-50 bp bovine coronavirus, a hepatitis D ribozyme sequence, a BGH poly(A) signal sequence, a CMV promoter sequence, and a 5'UTR sequence of 30-50 bp bovine coronavirus; the CMV promoter sequence is shown in SEQ ID NO: 7, the hepatitis D ribozyme sequence is shown in SEQ ID NO: 8, the BGH poly(A) signal sequence is shown in SEQ ID NO: 9, the 5'UTR sequence is shown in SEQ ID NO: 10, and the 3'UTR sequence is shown in SEQ ID NO: 11.

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