Bovine ephemeral fever virus strain and reverse genetic application thereof

Abstract

The invention belongs to the field of veterinary virology, and discloses a bovine ephemeral fever virus strain and reverse genetic application thereof, and the reverse genetic steps are as follows: taking pCI plasmid as a skeleton to prepare plasmid for expressing a complete genome of bovine ephemeral fever virus; the method comprises the following steps: respectively preparing auxiliary plasmids for expressing an N protein gene, a P protein gene, an L protein gene and a G protein gene of the bovine ephemeral fever virus by taking pCAGGS plasmids as a skeleton, co-transfecting BHK-21 cells by using the five plasmids, and culturing the cells to obtain the rescued bovine ephemeral fever virus. The rescued bovine ephemeral fever virus has no obvious difference from the reproductive capacity of the parent virus, and the genome is still stable after 10 generations of continuous passage in BHK-21 cells. The invention provides methodological support for genetic modification of the bovine ephemeral fever virus, and has an application prospect.

Description

Technical Field

[0001] This invention belongs to the field of veterinary virology and relates to a bovine ephemeral fever virus strain and its reverse genetics application. Background Technology

[0002] Bovine ephemeral fever (BEFV) is an acute, febrile, and highly contagious infectious disease caused by bovine ephemeral fever virus (BEFV). It primarily infects bovine animals, with clinical features including sudden onset of high fever, respiratory distress, joint pain, and lethargy. Although the mortality rate is relatively low, it can cause a sharp drop in milk production in dairy cows and stunted growth in beef cattle, resulting in severe economic losses to the livestock industry. This virus belongs to the genus *Transient Fever Virus* of the family Rhabdoviridae. It is a non-segmented, single-stranded, negative-sense RNA virus with a genome length of approximately 14.8 kb, encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase large protein (L). Among these, the G protein, as the virus's main immunogenic protein, contains multiple neutralizing antigenic sites and can induce protective humoral immunity, making it a core target for vaccine development.

[0003] Reverse genetics is a genetic method that studies gene function and its impact on phenotype by manipulating genes through site-directed mutations, insertions, or deletions. By artificially modifying viral genomic cDNA and then rescuing recombinant viruses from the cDNA clone, it is possible to precisely locate and modify viral genes, providing a core tool for viral gene function analysis, pathogenic mechanism research, and novel vaccine development. However, reverse genetics technology for bovine ephemeral fever virus (BOF) is not yet mature. Currently, there is a lack of stable and efficient whole-genome cDNA cloning and supporting helper plasmid systems, limiting in-depth research on its gene function and the development of novel vaccines. Therefore, constructing a stable and efficient reverse genetics system for BDF has become one of the key breakthroughs in current BDF prevention and control technology research. Summary of the Invention

[0004] Based on the problems existing in the prior art, this invention focuses on the BEFV reverse genetics system, optimizes and constructs the whole genome plasmid and auxiliary plasmid system of BEFVML3 strain, and establishes an efficient and stable BEFV reverse genetics operating system, breaking through the bottleneck of the prior art. This lays the foundation for in-depth analysis of the pathogenic mechanism of BEFV and the development of new vaccines and antiviral drugs, and is of great significance for improving the comprehensive prevention and control level of bovine ephemeral fever and ensuring the healthy development of animal husbandry.

[0005] To address the problems existing in the prior art, the first aspect of the present invention provides a plasmid combination, the plasmid combination including a first plasmid, a second plasmid, a third plasmid and a fourth plasmid;

[0006] The first plasmid contains a first gene expression cassette;

[0007] The second plasmid contains a second gene expression cassette;

[0008] The third plasmid contains a third gene expression cassette;

[0009] The fourth plasmid contains a fourth gene expression cassette;

[0010] The first gene expression cassette is capable of transcribing mRNA containing the coding sequence of the N protein of bovine ephemeral fever virus;

[0011] The second gene expression cassette is capable of transcribing mRNA containing the coding sequence of the P protein of bovine ephemeral fever virus;

[0012] The third gene expression cassette is capable of transcribing mRNA containing the coding sequence of the L protein of bovine ephemeral fever virus.

[0013] The fourth gene expression cassette is capable of transcribing mRNA containing the coding sequence of the G protein of bovine ephemeral fever virus.

[0014] In some embodiments, the plasmid assembly further includes a fifth plasmid containing a fifth gene expression cassette capable of transcribing bovine ephemeral fever virus genomic RNA (since negative-strand RNA viruses are named genomic RNA in positive-strand form, genomic RNA refers to positive-strand genomic RNA or positive-strand genomic RNA).

[0015] Plasmid combinations containing specific N, P, L, and G gene sequences (first, second, third, and fourth plasmids) can be used with transcripts of a fifth plasmid containing different genomic sequences to assemble rescued viruses. The genetic characteristics of the rescued virus in its passages depend on the genomic sequence in the fifth plasmid. Based on the research results of this invention, plasmid combinations of the first, second, third, and fourth plasmids possess relatively independent application value, demonstrating the technical concept of this invention.

[0016] In some embodiments, the backbone plasmid of the first plasmid is the pCAGGS plasmid;

[0017] The backbone plasmid of the second plasmid is the pCAGGS plasmid;

[0018] The backbone plasmid of the third plasmid is the pCAGGS plasmid;

[0019] The backbone plasmid of the fourth plasmid is the pCAGGS plasmid; or

[0020] The backbone plasmid of the fifth plasmid is the pCI plasmid.

[0021] In some implementations, the promoter in the first gene expression cassette is the T7 promoter;

[0022] In the second gene expression cassette, the promoter is the T7 promoter;

[0023] In the third gene expression cassette, the promoter is the T7 promoter;

[0024] In the fourth gene expression cassette, the promoter is the T7 promoter; or

[0025] The promoter in the fifth gene expression cassette is the T7 promoter.

[0026] In some embodiments, the amino acid sequence of the N protein of the bovine ephemeral fever virus is shown in SEQ ID NO.4;

[0027] The amino acid sequence of the P protein of the bovine ephemeral fever virus is shown in SEQ ID NO.5;

[0028] The amino acid sequence of the G protein of the bovine ephemeral fever virus is shown in SEQ ID NO.7;

[0029] The amino acid sequence of the L protein of the bovine ephemeral fever virus is shown in SEQ ID NO. 8; or

[0030] The genome of the bovine ephemeral fever virus is the sequence shown in SEQ ID NO.3 or the sequence shown in SEQ ID NO.3 with the T changed to C at position 9185.

[0031] A second aspect of the present invention provides a cell assembly comprising a first cell, a second cell, a third cell, and a fourth cell;

[0032] The first cell contains the first plasmid described in the first aspect of the present invention;

[0033] The second cell contains the second plasmid described in the first aspect of the present invention;

[0034] The third cell contains the third plasmid described in the first aspect of the present invention;

[0035] The fourth cell contains the fourth plasmid described in the first aspect of the present invention.

[0036] In some embodiments, the host cell of the first cell is selected from BHK-21 cells, BHK-T7 cells that stably express T7 RNA polymerase, and Escherichia coli cells;

[0037] The host cells for the second cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells;

[0038] The host cells of the third cell are selected from BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase, and Escherichia coli cells; or

[0039] The host cells of the fourth cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells.

[0040] A third aspect of the present invention provides a cell assembly comprising a first cell, a second cell, a third cell, a fourth cell, and a fifth cell;

[0041] The first cell contains the first plasmid described in the first aspect of the present invention;

[0042] The second cell contains the second plasmid described in the first aspect of the present invention;

[0043] The third cell contains the third plasmid described in the first aspect of the present invention;

[0044] The fourth cell contains the fourth plasmid described in the first aspect of the present invention;

[0045] The fifth cell contains the fifth plasmid described in the first aspect of the present invention.

[0046] In some embodiments, the host cell of the first cell is selected from BHK-21 cells, BHK-T7 cells that stably express T7 RNA polymerase, and Escherichia coli cells;

[0047] The host cells for the second cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells;

[0048] The host cells of the third cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells.

[0049] The host cells for the fourth cell were selected from BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase, and Escherichia coli cells; or

[0050] The host cells of the fifth cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells.

[0051] A fourth aspect of this invention provides a method for preparing a rescued bovine ephemeral fever virus, the method comprising the following steps:

[0052] S1: Bovine ephemeral fever virus susceptible cells are co-transfected with the first plasmid, second plasmid, third plasmid, fourth plasmid and fifth plasmid described in the first aspect of the present invention to obtain transfected cells;

[0053] The first plasmid, the second plasmid, the third plasmid, the fourth plasmid, and the fifth plasmid can all survive in the bovine ephemeral fever virus susceptible cells;

[0054] The first gene expression cassette, the second gene expression cassette, the third gene expression cassette, the fourth gene expression cassette, and the fifth gene expression cassette can all be expressed in the bovine ephemeral fever virus susceptible cells;

[0055] S2: Culture the transfected cells to obtain cultured cells; and

[0056] S3: The bovine ephemeral fever virus rescue virus is isolated from the cultured cells or the culture medium in which the cultured cells are cultured.

[0057] In some embodiments, the bovine ephemeral fever virus susceptible cells are BHK cells;

[0058] In S1, the molar ratio of the first plasmid, the second plasmid, the third plasmid, the fourth plasmid, and the fifth plasmid is 1:2-4:2-4:2-4:1-4;

[0059] In S1, the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid are mixed to obtain a plasmid mixture;

[0060] The plasmid mixture was mixed with the transfection reagent at a weight ratio of 1:1-4, and allowed to stand for 10-30 minutes to obtain the transfection complex.

[0061] The transfection complex was added to a culture monolayer of bovine ephemeral fever virus susceptible cells to obtain the transfected cells;

[0062] In S2, incubate at 35-39℃ for 3-5 days under conditions of 4-6% CO2; or

[0063] In S3, the cultured cells are frozen and thawed, and the supernatant is collected by centrifugation to obtain the bovine ephemeral fever virus rescue virus.

[0064] In some embodiments, the bovine ephemeral fever virus susceptible cells are BHK-21 cells that stably express T7 RNA polymerase; the promoter in the first gene expression cassette is the T7 promoter; the promoter in the second gene expression cassette is the T7 promoter; the promoter in the third gene expression cassette is the T7 promoter; the promoter in the fourth gene expression cassette is the T7 promoter; and the promoter in the fifth gene expression cassette is the T7 promoter.

[0065] In S1, the culture monolayer is prepared in a 6-well plate, and the total weight of the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid in the transfection complex added to each well is 1-3 μg.

[0066] In S1, the cell density of the culture monolayer is 80%-90%;

[0067] In S1, the culture medium for cultivating the monolayer is serum-free Opti-MEM medium; or

[0068] The transfection reagent is a PEI transfection reagent.

[0069] The fifth aspect of the present invention provides a bovine ephemeral fever virus rescue virus, which is prepared using the preparation method described in the fourth aspect of the present invention. Attached Figure Description

[0070] Figure 1 Photographs showing the identification of the isolated virus strain BEFV-ML3 using an indirect immunofluorescence method.

[0071] Figure 2 This is a schematic diagram of the genome sequence assembly strategy for the BEFV-ML3 strain.

[0072] Figure 3 Electrophoresis results of different steps in the splicing of the full-length genome of the BEFV-ML3 strain.

[0073] Figure 4 Electrophoresis results of the BEFV reverse genetics system helper plasmid.

[0074] Figure 5 Images showing the IFA identification results of four helper plasmids.

[0075] Figure 6 Photo of IFA identification results for BEFV virus rescue.

[0076] Figure 7 The growth curves of rBEFV ML3 and its parent are shown. Detailed Implementation

[0077] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0078] Example 1: Isolation and identification of bovine ephemeral fever virus (BEFV) ML3 strain

[0079] 1. Sample processing

[0080] Anticoagulated blood samples collected from cattle suspected of having bovine ephemeral fever (BEF) from a cattle farm in Henan Province were freeze-thawed three times, centrifuged to collect the supernatant, filtered through a 0.22 μm filter membrane for sterilization, and stored at -70℃ for later use.

[0081] 2. RT-PCR detection

[0082] Based on the BEFV G gene sequence published in Genbank, upstream primer BEFV-U (SEQ ID NO.1) and downstream primer BEFV-L (SEQ ID NO.2) were designed and synthesized in the conserved region of the G gene using Oligo 6.0.

[0083] RNA extraction was performed according to the instructions of the kit (purchased from Tiangen Biotech Co., Ltd.), obtaining RNA from the aforementioned filtrate sample. The reverse transcription system consisted of: 10 μL 5×PrimeScript Buffer, 6 μL dNTPs, 6 μL BEFV-L, 1 μL RRI, 1 μL PrimeScript, and 26 μL template RNA. The reverse transcription program was: 25℃ for 10 min, then 42℃ for 1 h. The obtained cDNA was used for subsequent PCR reactions. Using specific primers, the corresponding gene fragment of BEFV was amplified by RT-PCR. The PCR system consisted of: 10 μL 5×PrimeSTAR Buffer, 4 μL dNTPs, 2 μL BEFV-L, 2 μL BEFV-U, 0.5 μL PrimeSTAR, 29.5 μL ddH2O, and 2 μL cDNA. The procedure was as follows: 98℃ for 2 min, 98℃ for 10 s, 55℃ for 15 s, 72℃ for 1 min, 72℃ for 10 min, for 35 cycles. The amplified products were detected by 1% agarose gel electrophoresis, and the PCR products were detected by 1.0% agarose gel electrophoresis. The results showed that a specific band was amplified, consistent with the expected 420 bp size, indicating that the sample was BEFV positive.

[0084] 3. Virus isolation

[0085] The samples identified as positive by RT-PCR were inoculated into KC cells (Culicoidessonorensis KC, derived from Culicoides midge larvae, purchased from ATCC) at a ratio of 1:100 for primary culture. The cells were then placed in a 30°C, 5% CO2 incubator for 4-6 days. The virus was harvested and inoculated into BHK-21 cells that had grown into a monolayer. The cells were then passaged, and the virus was harvested for identification when cytopathic effects appeared.

[0086] 4. RT-PCR identification

[0087] The virus in the cells that showed cytopathic effects in step 3 was subjected to RT-PCR (RT-PCR method is the same as in step 2 above), and the results showed that the target band was amplified, which was consistent with the expected size.

[0088] 5. Identification by indirect immunofluorescence assay (IFA)

[0089] The virus harvested in step 3 was inoculated at a 1% ratio into a monolayer of well-grown BHK-21 cells and cultured at 37°C in a cell culture incubator containing 5% CO2 for 36 hours. The cell culture medium was discarded, and 100 μl of cold acetone fixative was added to each well. The cells were fixed for 15 minutes, the fixative was discarded, and the cells were allowed to air dry. 100 μl of PBS containing 5% skim milk was added to each well, and the cells were placed on a shaker and blocked at 37°C for 1 hour. The blocking solution was discarded, and the cells were washed three times with PBS and then dried. Add 100 μl of BEFV positive serum (serum obtained by centrifugation of whole blood collected from cattle immunized with BEFV inactivated vaccine) diluted 100 times with pH 7.4 PBS, incubate at 37°C for 1 hour, and wash 3 times with PBS for 3 minutes each time. Add 100 μl of FITC-labeled rabbit anti-bovine IgG (purchased from Sigma) diluted 100 times with pH 7.4 PBS, incubate at 37°C for 1 hour, wash 3 times with PBS for 3 minutes each time, and finally add 100 μl of PBS to each well and observe the results under a fluorescence microscope.

[0090] The virus harvested in step 3 was replaced with BEFV BEFV / HN3 / 2024 strain (the process of obtaining this strain is described in patent application CN202510742624.9, and the corresponding microbial accession number is CGMCC No.46379) as a positive control.

[0091] Parallel operations were performed using uninoculated BHK-21 cells as a negative control.

[0092] See results Figure 1As can be seen, the positive control wells exhibited specific green fluorescence, while the negative control wells did not. The presence of specific green fluorescence in the sample wells indicated that the tested samples were BEFV positive. Based on these results, RT-PCR and IFA identification confirmed that the isolated virus strain was bovine ephemeral fever virus (BEFV), and it was named BEFV-ML3 strain, or ML3 strain for short.

[0093] 6. Whole genome sequence analysis

[0094] High-throughput sequencing of the BEFV-ML3 strain was performed using a MGISEQ-200 next-generation sequencer manufactured by BGI Genomics, and the complete genome sequence was obtained by assembling the sequence using the accompanying software. In the field of virology, it is customary to record the genomes of negative-sense RNA viruses using the sequence of the positive-sense RNA. The genome sequence of the BEFV-ML3 strain recorded using the positive-sense RNA is shown in SEQ ID NO.3.

[0095] In the BEFV-ML3 genome, positions 1-88 are the 5' UTR (upstream of the positive strand), positions 89-1384 are the N protein coding sequence, positions 1432-2268 are the P protein coding sequence, positions 2337-3008 are the M protein coding sequence, positions 3075-4946 are the G protein coding sequence, positions 5021-6150 are the GNS protein coding sequence, positions 8420-14854 are the L protein coding sequence, and positions 14855-14934 are the 3' UTR (downstream of the positive strand).

[0096] The amino acid sequence of the N protein is shown in SEQ ID NO.4, the amino acid sequence of the P protein is shown in SEQ ID NO.5, the amino acid sequence of the M protein is shown in SEQ ID NO.6, the amino acid sequence of the G protein is shown in SEQ ID NO.7, and the amino acid sequence of the L protein is shown in SEQ ID NO.8.

[0097] Sequence analysis indicates that the strain is bovine ephemeral fever virus.

[0098] Example 2: Amplification of full-length bovine ephemeral fever virus (BEV) genomic cDNA and construction of recombinant vector pCI-BEFV

[0099] 1. Viral nucleic acid extraction

[0100] BHK-21 cell cultures infected with the BEFV-ML3 strain obtained in Example 1 were used to extract total viral RNA using the Trizol method. The RNA purity and integrity were tested to ensure that the A260 / A280 ratio was between 1.8 and 2.0.

[0101] 2. Gene fragment PCR amplification

[0102] Using the RNA extracted in step 1 as a template, reverse transcription was performed using oligo d(T) primers. PCR amplification was then performed on eight overlapping fragments (with 20 bp overlap between adjacent amplified fragments) covering the genome of strain BEFV-ML3 using PrimeSTAR high-fidelity DNA polymerase (TaKaRa). The eight primer pairs, in order from upstream to downstream according to their corresponding genomic positions, are PCI-BEFV-1F (SEQ ID NO.9) / Z-1R (SEQ ID NO.10); Z-2F (SEQ ID NO.11) / Z-2R (SEQ ID NO.12); Z-3F (SEQ ID NO.13) / Z-3R (SEQ ID NO.14); Z-4F (SEQ ID NO.15) / Z-4R (SEQ ID NO.16); Z-5F (SEQ ID NO.17) / Z-5R (SEQ ID NO.18); Z-6F (SEQ ID NO.19) / Z-6R (SEQ ID NO.20); Z-7F (SEQ ID NO.21) / Z-7R (SEQ ID NO.22); and Z-8F (SEQ ID NO.23) / BEFV-PCI-8R (SEQ ID NO.24). To further identify the rescue virus and the parent virus, a synonymous mutant molecular tag (corresponding to a T mutation to C at 9185nt) was introduced using primer Z-6F.

[0103] The RT reaction system consisted of 5×PSRT buffer 5 μl, dNTP Mix 2.5 μl, Primerscript 0.5 μl, oligo(T) 1 μl, and RNA 16 μl, for a total of 25 μl. The RT reaction program was 25℃ for 10 min and 42℃ for 60 min.

[0104] Using cDNA as a template, PCR amplification was performed using the aforementioned 8 pairs of primers. The PCR reaction system (50 μL) consisted of: 25 μL of 2×TaqPlus Master Mix, 1 μL each of forward and reverse primers (10 μmol / L), 2 μL of cDNA template, and sterile water added to a final volume of 50 μL. PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58-62℃ annealing for 30 s (adjusted according to primer Tm value), 72℃ extension for 1-2 min (adjusted according to fragment length), 35 cycles; and a final extension at 72℃ for 10 min. The amplified products were identified by 1% agarose gel electrophoresis, the target fragment was recovered, and the sequence of the amplified products was verified by Sanger sequencing before proceeding to the next step.

[0105] 3. Fragment splicing

[0106] The eight fragments obtained from PCR amplification were recovered from the gel and assembled using overlap extension PCR. A schematic diagram of the genome assembly scheme is shown below. Figure 2 First, four pairs of fragments, F1-F2, F3-F4, F5-F6, and F7-F8, were fused separately. Then, two pairs of fragments, F1-F2-F3-F4 and F5-F6-F7-F8, were fused. Finally, two large fragments were fused. Each fusion was performed using overlap extension PCR, specifically using the aforementioned fragment pairs and upstream primers from the upstream fragment and downstream primers from the downstream fragment shown in step 2. The PCR reaction was performed step by step to obtain the complete BEFV whole genome cDNA, with the viral genome sequence in the middle and homologous arm sequences for plasmid insertion upstream and downstream. After electrophoresis recovery, the spliced ​​product was verified by Sanger sequencing to ensure sequence accuracy before proceeding to the next step.

[0107] Electrophoresis results of different steps in the full-length genome assembly of BEFV-ML3 strain are shown in the images. Figure 3 Therefore, the splicing result meets expectations.

[0108] 4. Construction of recombinant vectors

[0109] The full-length cDNA fragment recovered in step 3 and the pCI plasmid (purchased from Promega) were digested with restriction endonucleases Nhe I and Not I, respectively. The digestion products were purified and ligated using the infusion method. The ligation product was transformed into DH5α competent *E. coli* cells, plated on LB agar plates containing ampicillin, and incubated at 37°C for 12–16 hours. Single colonies were picked, and the plasmid was extracted for restriction enzyme digestion identification and sequencing verification, confirming the correct construction of the recombinant vector (named pCI-BEFV).

[0110] The pCI-BEFV viral genome expression cassette has a T7 promoter and an SV40 Poly (A) terminator. The sequence that can be transcribed is the viral genome in the form of a single-stranded positive-sense RNA of the BEFV-ML3 strain, so as to interact with the proteins expressed by the helper plasmid to produce a rescued virus.

[0111] Example 3: Construction of the auxiliary plasmid system

[0112] 1. Target gene amplification

[0113] Using the genome of BEFV ML3 strain as a template, specific primer pairs pCAGGS-NF (SEQ ID NO.25) / pCAGGS-NR (SEQ ID NO.26); pCAGGS-PF (SEQ ID NO.27) / pCAGGS-PR (SEQ ID NO.28); pCAGGS-LF (SEQ ID NO.29) / pCAGGS-LR (SEQ ID NO.30); and pCAGGS-GF (SEQ ID NO.31) / pCAGGS-GR (SEQ ID NO.32) were designed for amplifying the coding regions of each gene using RT-PCR. Amplification conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1-3 min (N gene 1 min, G gene 1.5 min, P gene 1.5 min, L gene 3 min), for a total of 35 cycles; final extension at 72℃ for 10 min. PCR products were recovered by gel electrophoresis.

[0114] See electrophoresis results Figure 4 Therefore, it can be seen that the four genes were successfully amplified.

[0115] 2. Construction of auxiliary plasmids

[0116] The amplified N, P, L, and G gene fragments were digested with EcoRI by the pCAGGS plasmid (preserved by Harbin Veterinary Research Institute). The digestion products were purified and ligated using the infusion method, then transformed into DH5α competent cells. The cells were plated on LB agar containing ampicillin. Single colonies were picked the following day for colony PCR verification. Positive strains were sequenced using the Sanger sequencing method. Plasmids were extracted from the correctly sequenced bacterial cultures and named helper plasmids pCAGGS-N, pCAGGS-P, pCAGGS-G, and pCAGGS-L, respectively.

[0117] In the N, P, G, and L gene expression cassettes of the helper plasmids, the promoters were all T7 promoters and the terminators were all SV40polyA.

[0118] 3. Identification of helper plasmids

[0119] Four helper plasmids were identified using indirect immunofluorescence. BHK-T7 cells (BHK-21 cells stably expressing T7 RNA polymerase, self-made) were transfected with the helper plasmids in duplicate wells. Positive controls (infected with BEFV-ML3 virus strain) and negative controls (parallel experiments using empty pCAGGS vector) were also included. After 36 hours, the culture medium was discarded, and the cells were washed three times with PBST. 200 μl of pre-chilled 95% anhydrous ethanol was added to each well, and the cells were fixed at room temperature for 20 min. The anhydrous ethanol was discarded, and the cells were air-dried. BEFV-positive serum diluted 1:200 was added to each well. The cells were incubated at 37°C for 1 hour and washed three times with PBST. FITC-labeled rabbit anti-bovine IgG (1:200, purchased from Sigma) was added as a secondary antibody, and the cells were incubated at 37°C for 1 hour. The secondary antibody was discarded, and the cells were washed three times with PBST. Finally, 100 μl of PBS was added to each well, and the cells were observed using an inverted fluorescence microscope.

[0120] See results Figure 5 Therefore, pCAGGS-N, pCAGGS-P, pCAGGS-G, and pCAGGS-L can be normally expressed in BHK-T7 cells.

[0121] Example 4: Rescue and Identification of Recombinant Viruses

[0122] 1. Cell transfection

[0123] The recombinant plasmid pCI-BEFV encoding the full-length viral genome constructed in Example 2 and the four helper plasmids pCAGGS-N, pCAGGS-P, pCAGGS-G and pCAGGS-L constructed in Example 3 were co-transfected into BHK-T7 cells for initial virus rescue.

[0124] The specific steps are as follows:

[0125] (1) Preparation of monolayer cells

[0126] BHK-T7 cells cultured in 10% DMEM medium were transferred to 6-well plates. The next day, before transfection, the 10% DMEM medium in each well was replaced with 1 mL of preheated 37°C serum-free Opti-MEM medium to obtain a BHK-T7 cell monolayer.

[0127] (2) Preparation of transfection complex

[0128] The plasmids pCI-BEFV, pCAGGS-N, pCAGGS-P, pCAGGS-G, and pCAGGS-L used for transfection were all diluted with PBS to a concentration of 0.1 μg / μl beforehand. A mixture of pCI-BEFV, pCAGGS-N, pCAGGS-P, pCAGGS-G, and pCAGGS-L plasmids was prepared at a mass ratio of 2:1:1:1:1, at a concentration of 2 μg / well (calculated based on the total weight of the five plasmids). The PEI transfection reagent was thoroughly mixed with the plasmids at a volume-to-mass ratio of 3 ml:1 g, and allowed to stand at room temperature for 15 min for transfection complex preparation.

[0129] (3) Plasmid transfection

[0130] Add the transfection complex dropwise to the culture medium of the above monolayer BHK-T7 cells, gently shake the cell plate to mix, and incubate at 37°C in a 5% CO2 incubator for 3-5 days.

[0131] 2. Virus rescue and identification

[0132] The transfected cells were cultured for another 3-5 days, and the viral culture was harvested. The culture was then frozen and thawed twice, and the supernatant (viral fluid) was collected by centrifugation. This supernatant was then passaged into BHK-T7 cells for identification. The rescued virus was named rBEFV ML3, or rBEFV for short.

[0133] The third-generation rescue virus was identified, including one-step growth curves, IFA, and molecular tag identification.

[0134] Virus-specific genes were detected using RT-PCR with primers Z-5F and Z-6R. Electrophoresis results showed that rBEFV could amplify the target band, and sequencing results showed that it possessed an artificially designed molecular tag (9185nt T mutation to C). The rescue virus rBEFV ML3 and the parent virus BEFV-ML3 had similar CPE characteristics. Viral protein expression was detected by indirect immunofluorescence assay (method as in step 5 of Example 1), and specific fluorescence was observed in the cells. Figure 6 ).

[0135] Viral titer assays showed that rBEFV titers could reach 10. 6 TCID 50 / mL, with no significant difference from wild-type strains, exhibiting consistent replication dynamics ( Figure 7 This indicates that the recombinant virus was successfully rescued.

[0136] 3. Optimization of conditions for virus rescue

[0137] Using the rescue efficiency and viral titer of recombinant virus rBEFV as evaluation indicators, the plasmid ratio and transfection conditions were optimized.

[0138] (1) Plasmid ratio optimization: Five different mass ratios of recombinant vector to helper plasmid (pCI-BEFV:pCAGGS-N:pCAGGS-P:pCAGGS-G:pCAGGS-L) were set as follows: 1:1:1:1:1, 1:1:1:1:2, 1:1:1:2:2, 1:2:1:1:1:2, and 2:1:1:1:2. Transfection was performed under the same conditions as in step 1. The results showed that when the ratio was 2:1:1:1:2, the recombinant virus rescue positivity rate reached 100%, and the viral titer was the highest (102). 6 TCID 50 The ratio of pCI-BEFV to pCAGGS-N to pCAGGS-P to pCAGGS-G to pCAGGS-L was significantly higher than that of other groups, and this ratio was determined to be the optimal plasmid ratio. When the plasmid weight ratio was 2:1:1:1:2, the molar ratio of pCI-BEFV:pCAGGS-N:pCAGGS-P:pCAGGS-G:pCAGGS-L was approximately 16:28:31:25:28.

[0139] (2) Optimization of transfection conditions: The effects of the volume ratio of transfection reagent to plasmid mixture (1:1, 2:1, 3:1) and room temperature incubation time (10 min, 20 min, 30 min) on the rescue effect were compared. The results showed that the cell transfection efficiency was highest, the virus rescue cycle was shortest (3-5 days), and the virus titer was stable after rescue when the cell density was 80%-90%, the volume ratio of transfection reagent to plasmid mixture was 2:1, and the incubation time was 20 min at room temperature. These were the optimal transfection conditions. In summary, the optimal rescue conditions for the recombinant virus of this invention are: cell density 80%-90%, plasmid molar ratio of approximately 16:28:31:25:28, transfection reagent to plasmid mixture volume ratio of 2:1, and incubation time at room temperature for 20 min.

[0140] 4. Identification of genetic stability of recombinant viruses

[0141] The genetic stability of the rescued recombinant virus (rBEFV ML3) was determined by passage 10 times in BHK-21 cells, with samples taken every two passages. Key functional genes (N, G, P, L) of the virus were amplified by RT-PCR. Sequencing results showed that the viral gene sequences of each passage were identical to the original rescued virus, with no base mutations, deletions, or insertions. Cytopathic effects were observed, with typical cytopathic effects such as rounding and shedding appearing after infection in each passage. Viral titer assays showed that the viral titer remained at 10 after continuous passage. 5 -10 6 TCID 50 The viral density was / mL, showing no significant difference from the parent virus. These results confirm that the recombinant virus rescued by this invention has stable genetic characteristics, and its replication characteristics did not change significantly after continuous passage, meeting the application needs of subsequent gene function research, vaccine development, and drug screening.

[0142] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A plasmid combination, the plasmid combination comprising a first plasmid, a second plasmid, a third plasmid, and a fourth plasmid; The first plasmid contains a first gene expression cassette; The second plasmid contains a second gene expression cassette; The third plasmid contains a third gene expression cassette; The fourth plasmid contains a fourth gene expression cassette; The first gene expression cassette is capable of transcribing mRNA containing the coding sequence of the N protein of bovine ephemeral fever virus; The second gene expression cassette is capable of transcribing mRNA containing the coding sequence of the P protein of bovine ephemeral fever virus; The third gene expression cassette is capable of transcribing mRNA containing the coding sequence of the L protein of bovine ephemeral fever virus. The fourth gene expression cassette is capable of transcribing mRNA containing the coding sequence of the G protein of bovine ephemeral fever virus.

2. The plasmid combination as described in claim 1, characterized in that, The plasmid assembly also includes a fifth plasmid containing a fifth gene expression cassette, which is capable of transcribing the genomic RNA of bovine ephemeral fever virus.

3. The plasmid combination as described in claim 2, characterized in that, The backbone plasmid of the first plasmid is the pCAGGS plasmid; The backbone plasmid of the second plasmid is the pCAGGS plasmid; The backbone plasmid of the third plasmid is the pCAGGS plasmid; The backbone plasmid of the fourth plasmid is the pCAGGS plasmid; or The backbone plasmid of the fifth plasmid is the pCI plasmid.

4. The plasmid combination as described in claim 2, characterized in that, In the first gene expression cassette, the promoter is the T7 promoter; In the second gene expression cassette, the promoter is the T7 promoter; In the third gene expression cassette, the promoter is the T7 promoter; In the fourth gene expression cassette, the promoter is the T7 promoter; or The promoter in the fifth gene expression cassette is the T7 promoter.

5. The plasmid combination according to any one of claims 2-4, characterized in that, The amino acid sequence of the N protein of the bovine ephemeral fever virus is shown in SEQ ID NO.4; The amino acid sequence of the P protein of the bovine ephemeral fever virus is shown in SEQ ID NO.5; The amino acid sequence of the G protein of the bovine ephemeral fever virus is shown in SEQ ID NO.7; The amino acid sequence of the L protein of the bovine ephemeral fever virus is shown in SEQ ID NO. 8; or The genome of the bovine ephemeral fever virus is the sequence shown in SEQ ID NO.3 or the sequence shown in SEQ ID NO.3 with the T changed to C at position 9185.

6. A cell assembly comprising a first cell, a second cell, a third cell, and a fourth cell; The first cell contains the first plasmid as described in claim 1, 3, 4 or 5; The second cell contains the second plasmid as described in claim 1, 3, 4 or 5; The third cell contains the third plasmid as described in claim 1, 3, 4 or 5; The fourth cell contains the fourth plasmid as described in claim 1, 3, 4 or 5.

7. The cell combination as described in claim 6, characterized in that, The host cells of the first cell were selected from BHK-21 cells, BHK-T7 cells that stably express T7 RNA polymerase, and Escherichia coli cells; The host cells for the second cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells; The host cells of the third cell are selected from BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase, and Escherichia coli cells; or The host cells of the fourth cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells.

8. A cell assembly comprising a first cell, a second cell, a third cell, a fourth cell, and a fifth cell; The first cell contains the first plasmid as described in any one of claims 2-5; The second cell contains the second plasmid as described in any one of claims 2-5; The third cell contains the third plasmid as described in any one of claims 2-5; The fourth cell contains the fourth plasmid as described in any one of claims 2-5; The fifth cell contains the fifth plasmid as described in any one of claims 2-5.

9. The cell assembly as described in claim 8, characterized in that, The host cells of the first cell were selected from BHK-21 cells, BHK-T7 cells that stably express T7 RNA polymerase, and Escherichia coli cells; The host cells for the second cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells; The host cells of the third cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells. The host cells for the fourth cell were selected from BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase, and Escherichia coli cells; or The host cells of the fifth cell were selected from BHK-21 cells, BHK-21 cells that stably express T7 RNA polymerase, and Escherichia coli cells.

10. A method for preparing a rescued bovine ephemeral fever virus, the method comprising the following steps: S1: Co-transfect bovine ephemeral fever virus susceptible cells with the first plasmid, second plasmid, third plasmid, fourth plasmid and fifth plasmid as described in any one of claims 2-5 to obtain transfected cells; The first plasmid, the second plasmid, the third plasmid, the fourth plasmid, and the fifth plasmid can all survive in the bovine ephemeral fever virus susceptible cells; The first gene expression cassette, the second gene expression cassette, the third gene expression cassette, the fourth gene expression cassette, and the fifth gene expression cassette can all be expressed in the bovine ephemeral fever virus susceptible cells; S2: Culture the transfected cells to obtain cultured cells; S3: The bovine ephemeral fever virus rescue virus is isolated from the cultured cells or the culture medium in which the cultured cells are cultured.

11. The preparation method according to claim 10, characterized in that, The susceptible cells for bovine ephemeral fever virus were BHK cells; In S1, the molar ratio of the first plasmid, the second plasmid, the third plasmid, the fourth plasmid, and the fifth plasmid is 1:2-4:2-4:2-4:1-4; In S1, the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid are mixed to obtain a plasmid mixture; The plasmid mixture was mixed with the transfection reagent at a weight ratio of 1:1-4, and allowed to stand for 10-30 minutes to obtain the transfection complex. The transfection complex was added to a culture monolayer of bovine ephemeral fever virus susceptible cells to obtain the transfected cells; In S2, incubate at 35-39℃ for 3-5 days under conditions of 4-6% CO2; or In step S3, the cultured cells are frozen and thawed, and the supernatant is collected by centrifugation to obtain the bovine ephemeral fever virus rescue virus.

12. The preparation method according to claim 11, characterized in that, The bovine ephemeral fever virus susceptible cells are BHK-21 cells that stably express T7 RNA polymerase; the promoter in the first gene expression cassette is the T7 promoter; the promoter in the second gene expression cassette is the T7 promoter; the promoter in the third gene expression cassette is the T7 promoter; the promoter in the fourth gene expression cassette is the T7 promoter; and the promoter in the fifth gene expression cassette is the T7 promoter. In S1, the culture monolayer is prepared in a 6-well plate, and the total weight of the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid in the transfection complex added to each well is 1-3 μg. In S1, the cell density of the culture monolayer is 80%-90%; In S1, the culture medium for cultivating the monolayer is serum-free Opti-MEM medium; or The transfection reagent is a PEI transfection reagent.

13. A bovine ephemeral fever virus rescue virus, wherein the bovine ephemeral fever virus rescue virus is prepared by the preparation method according to any one of claims 10-12.

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