Chimeric swine fever virus, swine fever marker vaccine, and preparation method and application thereof

Abstract

The present application discloses a chimeric classical swine fever virus, a classical swine fever marker vaccine, and a preparation method and application thereof. In the embodiment of the present application, the non-coding region and the main antigenic region of the E2 protein of the attenuated live classical swine fever vaccine strain C were replaced with the corresponding region of the BVDV Hubei strain by reverse genetic manipulation technology, and an infectious clone of the chimeric classical swine fever virus was constructed. The infectious clone was rescued by cell transfection and passage to obtain a chimeric classical swine fever virus with stable characteristics. The chimeric classical swine fever virus can stably replicate in pig-derived cells and has genetic stability and safety; it can induce the production of specific antibodies after immunization of the body, and can also provide the pig with complete protection against lethal infection by the virulent classical swine fever virus. In addition, the strain can immunize to produce BVDV E2-specific antibodies to distinguish between vaccinated pigs and wild-type classical swine fever virus-infected pigs, so that the strain has an application prospect as a classical swine fever marker vaccine.

Description

Technical Field

[0001] The present application relates to the technical field of classical swine fever virus, specifically chimeric classical swine fever virus, classical swine fever marker vaccine and preparation method and application thereof. Background Art

[0002] Classical swine fever (CSF) is a highly contagious viral disease of pigs caused by infection with the classical swine fever virus (CSFV). CSFV is a virus with an envelope. The virus particles are slightly round with a diameter of 34-50 nanometers and an inner core diameter of about 30 nanometers. The surface of the virus particles has fragile fibrillar structures. The envelope is formed by a double layer of lipids and the structural glycoprotein E rns , E1 and E2 are embedded in the membrane, and the core capsid protein Core in the envelope is a regular dodecahedron that wraps the viral genome RNA.

[0003] Classical swine fever virus is a single-stranded positive-strand RNA virus with a genome length of approximately 12.3 kb, consisting of a large open reading frame (ORF) and two untranslated regions (UTRs). The 5'UTR does not contain a cap structure, and the 3'UTR does not have a poly A tail. The large ORF in the middle encodes a polyprotein of approximately 3898 amino acids (aa). After cleavage and processing by cellular or viral proteases, the polyprotein forms 12 mature proteins, including four structural proteins, Core, E rns (E0), E1, E2 and 8 nonstructural proteins N pro , p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B. Among them, the UTRs at both ends of the CSFV genome are important regulatory factors for viral RNA replication and translation, and the structural protein E2 is the main antigenic protein that can induce the production of specific antibodies and play a role in protecting against CSFV.

[0004] Vaccination is a powerful tool for protecting against CSF infection. Classical Chinese Fever Virus C strain, a rabbit-based live attenuated vaccine, is widely used in China and provides complete protection against infection with virulent CSFV strains. However, as a live attenuated vaccine, a major limitation is the inability to serologically distinguish vaccinated pigs from those infected with wild-type CSFV (DIVA), a significant hindrance to CSF decontamination. Summary of the Invention

[0005] To this end, the present application discloses a chimeric classical swine fever virus, a classical swine fever marker vaccine, and its preparation method and application. Reverse genetic manipulation technology is used to replace the non-coding regions (untranslated regions, UTRs) and the main antigenic region (aa 690-860) of the classical swine fever vaccine C-strain (C-strain) with the corresponding regions of the bovine viral diarrhea virus (BVDV) Hubei strain, and a chimeric classical swine fever virus infectious polynucleotide pC / bUTRs-tE2 is constructed. By transfecting cells with the infectious polynucleotide pC / bUTRs-tE2 and rescuing them through passage, an attenuated, stable chimeric classical swine fever virus rC / bUTRs-tE2, as well as a composition and a marker vaccine containing it as the main active ingredient are obtained. The chimeric swine fever virus rC / bUTRs-tE2 not only has stable genetic and biological characteristics and can replicate stably, but is also safe; it can induce the production of specific antibodies in the body and protect pigs against infection with virulent swine fever virus; and it can induce the production of distinguishable BVDV E2-specific antibodies to distinguish vaccinated pigs from wild swine fever virus-infected pigs, and has application prospects as a swine fever marker vaccine.

[0006] To this end, the embodiments of the present application at least disclose the following technical solutions:

[0007] In a first aspect, the present invention discloses an isolated infectious polynucleotide or infectious clone, wherein the infectious polynucleotide or the infectious clone carries a chimeric genome, wherein the chimeric genome comprises an open reading frame and a 5'UTR region and a 3'UTR region located at both ends of the open reading frame;

[0008] The nucleotide sequences of the 5'UTR region and the 3'UTR region are shown in SEQ ID NOs. 1 to 2, respectively;

[0009] The development reading frame encoding Core, E rns (E0), E1, E2, N pro ,p7,NS2,NS3,NS4A,NS4B,NS5A and NS5B;Core,E rns (E0), E1, N pro The nucleotide sequences of the genes encoding the C-terminal region of p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B, and E2 were all derived from the genome of the C strain of classical swine fever vaccine;

[0010] The nucleotide sequence encoding the N-terminal antigenic region of the E2 protein is shown in SEQ ID NO.3;

[0011] The infectious polynucleotide is introduced into cells and infectious classical swine fever virus particles can be produced through passaging.

[0012] In a second aspect, the embodiments of the present application disclose a vector comprising the infectious polynucleotide or the infectious clone described in the first aspect.

[0013] In a third aspect, the embodiments of the present application disclose a cell carrying the infectious polynucleotide or the infectious clone described in the first aspect.

[0014] In a fourth aspect, the present invention discloses a swine fever virus particle, wherein the swine fever virus particle encapsulates a single-stranded RNA carrying a chimeric genome, wherein the chimeric genome comprises an open reading frame and a 5'UTR region and a 3'UTR region at both ends of the open reading frame; the nucleotide sequences of the 5'UTR region and the 3'UTR region are shown in SEQ ID NOs. 1 and 2, respectively; the open reading frame encodes Core, E rns (E0), E1, E2, N pro ,p7,NS2,NS3,NS4A,NS4B,NS5A and NS5B;Core,E rns (E0), E1, N pro The nucleotide sequences of the genes encoding the C-terminal region of p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B and E2 are all from the genome of the C strain of classical swine fever vaccine; the nucleotide sequence encoding the N-terminal antigen region of the E2 protein is shown in SEQ ID NO.3.

[0015] In a fifth aspect, the present application discloses a composition or a labeled vaccine, comprising classical swine fever virus particles, wherein the classical swine fever virus particles encapsulate a single-stranded RNA carrying a chimeric genome, wherein the chimeric genome comprises an open reading frame and a 5'UTR region and a 3'UTR region at both ends of the open reading frame; the nucleotide sequences of the 5'UTR region and the 3'UTR region are shown in SEQ ID NOs. 1 to 2, respectively; the open reading frame encodes Core, E rns (E0), E1, E2, N pro ,p7,NS2,NS3,NS4A,NS4B,NS5A and NS5B;Core,E rns (E0), E1, N pro The nucleotide sequences of the genes encoding the C-terminal region of p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B and E2 are all from the genome of the C strain of classical swine fever vaccine; the nucleotide sequence encoding the N-terminal antigen region of the E2 protein is shown in SEQ ID NO.3.

[0016] In a sixth aspect, the embodiments of the present application disclose the use of the infectious polynucleotide or infectious clone described in the first aspect in the preparation of a swine fever marker vaccine.

[0017] Compared with the prior art, this application has at least the following effective effects:

[0018] The embodiments of the present application construct a chimeric classical swine fever virus, a marker vaccine thereof, a preparation method thereof and an application thereof. One of the embodiments provides an infectious clone pC / bUTRs-tE2 of the constructed chimeric attenuated classical swine fever vaccine C strain, and the attenuated, stably passaged chimeric classical swine fever virus C strain rC / bUTRs-tE2 and a vaccine with it as the main active ingredient and its composition are obtained by transfecting cells and rescuing through passage. The chimeric attenuated live vaccine rC / bUTRs-tE2 can not only replicate stably, but also has safety; it can induce the production of specific antibodies in the body and can also protect pigs against virulent infection of classical swine fever virus; and it can induce the production of distinguishable BVDV E2-specific antibodies to distinguish between vaccinated pigs and wild-type classical swine fever virus-infected pigs, and has application prospects as a marker vaccine for classical swine fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the genome construction of the chimeric classical swine fever virus rC / bUTRs-tE2 provided in the examples of this application.

[0020] Figure 2 Schematic diagram of the structure of the infectious clone pC / bUTRs-tE2 of the chimeric classical swine fever virus provided in the examples of the present application, which includes the 5'UTR region and 3'UTR region from BVDV, and the E2 antigen region from BVDV (GenBank: MZ484396).

[0021] Figure 3 This is a gel electrophoresis result of the restriction enzyme fragment of the infectious clone pC / bUTRs-tE2 of the chimeric swine fever virus provided in the embodiment of the present application. The names of the electrophoresis bands are indicated above the figure, and the 9208bp and 5890bp are indicated on the right. Figure 2 The target fragment of pC / bUTRs-tE2 was double-digested with BamHI and MluI.

[0022] Figure 4 This is an immunofluorescence staining image of PK15 cells transfected with chimeric classical swine fever virus rC / bUTRs and rC / bUTRs-tE2 provided in the examples of this application.

[0023] Figure 5 This is a graph showing the RT-PCR results of the supernatant of PK15 cells rescued after transfection of the chimeric classical swine fever virus rC / bUTRs and rC / bUTRs-tE2 provided in the examples of this application.

[0024] Figure 6 These are immunofluorescence staining images of PK15 cells infected with P0, P10, P20, P30 and P40 of the chimeric classical swine fever virus rC / bUTRs-tE2 provided in the examples of this application.

[0025] Figure 7 This is a graph showing the viral titer results of P0, P10, P20, P30 and P40 of the chimeric classical swine fever virus rC / bUTRs-tE2 provided in the examples of the present application on PK15 cells.

[0026] Figure 8 These are immunofluorescence staining images of PK15 cells and MDBK cells infected with the classical swine fever virus C strain, rC / bUTRs-tE2, and rC / bUTRs provided in the examples of this application, respectively.

[0027] Figure 9 This is a graph showing the results of genomic RNA replication of classical swine fever virus strain C, rC / bUTRs-tE2, and rC / bUTRs in PK15 cells provided in the examples of this application.

[0028] Figure 10 The plaques formed by the classical swine fever virus C strain, rC / bUTRs-tE2 and rC / bUTRs on SK6 cells (left figure) and the size statistical results (right figure) provided in the examples of this application.

[0029] Figure 11 Figures showing the antibody production results in rabbits induced by Classical Swine Fever Virus strain C, rC / bUTRs-tE2, and rC / bUTRs, respectively, as provided in the examples of this application. Left: CSFV-specific antibodies induced by rC / bUTRs-tE2 in rabbits. CSFV-specific antibodies were detected using the IDEXX commercial CSFV-specific antibody kit. A blocking value of ≥40% was considered positive (cut-off line). Right: Virus-neutralizing antibody titers induced by rC / bUTRs-tE2 in rabbits. Serum virus-neutralizing antibody titers were measured using serum neutralization experiments against the virulent Shimen strain of CSFV.

[0030] Figure 12 This is a rectal temperature chart of pigs immunized with the Classical Swine Fever Virus C strain and rC / bUTRs-tE2 provided in the examples of this application.

[0031] Figure 13The clinical symptom scores and survival curves of pigs after immunization and infection with the classical swine fever virus strain C and rC / bUTRs-tE2 (DMEM as a control) provided in the examples of this application. Left figure: Clinical symptom scoring of pigs. All pigs were scored according to the classical swine fever clinical scoring standard established by Mittelholzer et al., including vitality, body tension, body shape, breathing, walking, skin (especially ears, nose, legs and tail), eyes / conjunctiva, appetite, defecation and residual feed. Normal clinical symptoms are 0 points, mild clinical symptoms are 1 point, obvious clinical symptoms are 2 points, severe clinical symptoms are 3 points, and all scores are added up to a maximum clinical symptom score of 30 points. Right figure: Survival curve of pigs after infection.

[0032] Figure 14 White blood cell and platelet counts in pigs after immunization and challenge with Classical Swine Fever virus strain C and rC / bUTRs-tE2 (DMEM as a control) as provided in the examples of this application. Left: White blood cell counts in pigs after immunization and challenge. Right: Platelet counts in pigs after immunization and challenge.

[0033] Figure 15 The results of CSFV-specific antibody and virus-neutralizing antibody titers in pigs after immunization and challenge with the classical swine fever virus C strain and rC / bUTRs-tE2 (DMEM as a control) provided in the examples of this application. Left: CSFV-specific antibodies induced in pigs by the classical swine fever vaccine C strain non-coding region and E2-replaced recombinant chimeric virus vaccine strain rC / bUTRs-tE2. CSFV-specific antibodies were detected by IDEXX's commercial CSFV-specific antibody kit, and an antibody blocking value of ≥40% was considered positive. Right: Virus-neutralizing antibody titers induced in pigs by the classical swine fever vaccine C strain non-coding region and E2-replaced recombinant chimeric virus vaccine strain rC / bUTRs-tE2. Serum neutralizing antibody titers were measured by serum neutralization experiments with the virulent Shimen strain of CSFV.

[0034] Figure 16 The CSFV E provided in the embodiment of this application rns Results of indirect ELISA detection of recombinant chimeric virus vaccine strain rC / bUTRs-tE2 immune serum as coating antigen. Left: CSFV E rns Indirect ELISA detection of recombinant chimeric virus vaccine strain rC / bUTRs-tE2 immune rabbit serum. Right: CSFV E rns Indirect ELISA was used to detect the serum of pigs immunized with the recombinant chimeric virus vaccine strain rC / bUTRs-tE2.

[0035] Figure 17Results of an indirect ELISA assay using BVDV tE2 as the coating antigen for sera from the recombinant chimeric virus vaccine strain rC / bUTRs-tE2, as provided in the examples of this application. Left: BVDV tE2 indirect ELISA assay for rabbit sera immunized with the recombinant chimeric virus vaccine strain rC / bUTRs-tE2. Right: BVDV tE2 indirect ELISA assay for sera from pigs immunized with the recombinant chimeric virus vaccine strain rC / bUTRs-tE2.

[0036] Figure 18 CSFV E provided in the embodiment of this application rns Electrophoresis identification results of expression vector digestion products. The lanes are DL5000 marker, pET-28a-E rns Plasmid, Ncol I single digestion product, Xho I single digestion product and double digestion product, 5231bp is the vector band, 689bp is the target band.

[0037] Figure 19 CSFV E provided in the embodiment of this application rns The target protein was induced and identified by SDS-PAGE (left) and Western-Blot (right). rns SDS-PAGE identification of the induced expression of the target protein. M: marker; Lane 1: BL21 / pET28a uninduced whole bacteria; Lane 2: BL21 / pET28a induced whole bacteria; Lane 3: BL21 / pET-28a-E rns Uninduced whole bacteria; Lane 4: BL21 / pET-28a-E rns Induced whole bacteria; Lane 5: BL21 / pET-28a-E rns Induction supernatant; Lane 6: BL21 / pET-28a-E rns Induced precipitation. Right: CSFV E rns Western-blot analysis of the induced expression of the target protein. M: marker; Lane 1: BL21 / pET28a uninduced; Lane 2: BL21 / pET28a induced whole bacteria; Lane 3: BL21 / pET-28a-E rns Uninduced whole bacteria; Lane 4: BL21 / pET-28a-E rns Induced whole bacteria; Lane 5: BL21 / pET-28a-E rns Induction supernatant; Lane 6: BL21 / pET-28a-E rns Induce precipitation.

[0038] Figure 20 CSFV E provided in the embodiment of this application rnsSDS-PAGE analysis of the target protein eluate. M: marker; Lane 1: supernatant after binding; Lanes 2-4: contaminant protein eluate; Lanes 5-14: target protein eluate.

[0039] Figure 21 CSFV E provided in the embodiment of this application rns SDS-PAGE identification results after target protein renaturation. M: Protein marker; Lane 1: Whole cell without induction with BL21 / pET28a; Lane 2: Whole cell with induction with BL21 / pET28a; Lane 3: Whole cell without induction with BL21 / pET-28a-Erns; Lane 4: Whole cell with induction with BL21 / pET-28a-Erns; Lane 5: CSFV E after renaturation by dialysis rns . DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0041] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence, nor do they play a substantial limiting role on the subsequent technical features. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0042] Infectious polynucleotide or infectious clone

[0043] The present application examples disclose infectious polynucleotides and / or infectious clones of classical swine fever virus (CFSV). The term "infectious clone" as used herein is a polynucleotide having two or more nucleotides, which refers to a polynucleotide sequence capable of replicating in a prokaryotic host cell or a eukaryotic cell, and is used interchangeably with "infectious polynucleotide" herein. When an RNA polynucleotide is obtained by in vitro transcription and introduced into a receptive cell, it can replicate in the cell as RNA and produce infectious virus particles. Therefore, the chimeric classical swine fever virus genome can be present in a vector in the form of DNA, in a virus particle in the form of RNA, or in the form of isolated DNA or RNA. The term "polynucleotide" refers to a polymer of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides, including double-stranded and single-stranded DNA and RNA. Unless otherwise indicated, a polynucleotide includes its complementary form. Those skilled in the art can easily determine the complementary nucleotide sequence of a polynucleotide. A polynucleotide may include nucleotide sequences with different functions, including, for example, coding sequences and non-coding sequences (e.g., regulatory sequences and / or untranslated regions). Polynucleotides can be obtained directly from natural sources, or prepared by means of recombinant technology, enzymatic technology, or chemical technology. A polynucleotide may be linear or circular in topology.A polynucleotide may be, for example, a component or fragment of a vector (eg, an expression vector or a cloning vector).

[0044] If naturally occurring, a polynucleotide is preferably "isolated," and more preferably purified. An "isolated" compound (e.g., a polynucleotide, protein, or viral particle) is one that is separated and isolated from its natural environment. A "purified" compound is at least 60% free from other components with which it is naturally associated, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated. Compounds such as polynucleotides and polypeptides that are produced outside of a native organism (e.g., by chemical or recombinant methods) are, by definition, considered isolated and purified because they do not occur in their natural environment.

[0045] The present application embodiment discloses an isolated infectious polynucleotide or infectious clone, which carries at least one chimeric genome. In some embodiments, the infectious polynucleotide or infectious clone can carry multiple copies of the chimeric genome to enhance its infectivity or virus rescue efficiency. The chimeric genome comprises a large open reading frame (ORF) and untranslated regions (UTRs) at both ends. The ORF encodes a polyprotein of approximately 3898 amino acids (aa). After cleavage and processing by cell or viral proteases, the polyprotein forms 12 mature proteins, including 4 structural proteins Core, E rns(E0), E1, E2 and 8 nonstructural proteins N pro , p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B. Among them, the full length of the chimeric genome is a polynucleotide of about 12.3kb, the 5'UTR region is located in the 1-375nt range, the 3' UTR region is located in the 12068-12300nt range, and the coding region of the E2 protein is located in the 2441-3559nt range. Among them, the 5'UTR region is derived from bovine viral diarrhea virus (bovine viral diarrhea virus, BVDV Hubei strain), as shown in SEQ ID NO.1. The 3'UTR region is derived from the BVDV Hubei strain, as shown in SEQ ID NO.2. The antigenic region of the E2 protein coding region (as shown in SEQ ID NO.3) is derived from the region corresponding to the E2 protein coding region of the BVDV Hubei strain (GenBank accession number: MZ484396). Among them, Core, E rns (E0), E1, N pro The nucleotide sequences of the genes encoding the C-terminal region of p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B and E2 were all derived from the genome of the classical swine fever vaccine strain C (GenBank accession number AY805221).

[0046] In one embodiment, the CSFV vaccine C strain cDNA infectious clone pSPT Ⅰ Based on strain C, a chimeric classical swine fever virus cDNA infectious clone, pC / bUTRs, was constructed. pC / bUTRs carries a single copy of a chimeric genome derived from strain C, in which the UTRs at either end of the genome of strain C were replaced with the corresponding regions of BVDV. In this example, pC / bUTRs was transfected and passaged into cells to produce a chimeric classical swine fever virus, rC / bUTRs; compared to strain C, the chimeric classical swine fever virus rC / bUTRs exhibited significantly increased replication efficiency.

[0047] Furthermore, in one embodiment, another CSFV vaccine C strain cDNA infectious clone, pC / bUTRs-tE2, was constructed. pC / bUTRs-tE2 carries a chimeric genome in which the UTRs at both ends of the C strain genome are replaced with regions corresponding to BVDV, and the N-terminus of the E2 protein (amino acids 690-860, the main antigenic region) is replaced with a region corresponding to BVDV. By transfecting cells with the cDNA infectious clone pC / bUTRs-tE2 and passaged, a chimeric classical swine fever virus strain pC / bUTRs-tE2 can be rescued. The chimeric swine fever virus pC / bUTRs-tE2 not only has stable genetic and biological characteristics and can replicate stably, but is also safe; it can induce the production of specific antibodies in the body and protect pigs against infection with virulent swine fever virus; and it can induce the production of distinguishable BVDV E2-specific antibodies to distinguish vaccinated pigs from wild swine fever virus-infected pigs, and has application prospects as a swine fever marker vaccine.

[0048] "Vector" refers to a recombinant DNA or RNA plasmid or virus containing a heterologous polynucleotide to be delivered to a target cell in vitro or in vivo. For prophylactic or therapeutic purposes, the heterologous polynucleotide may contain a target sequence and may optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.

[0049] The terms "nucleic acid" and "nucleic acid sequence" refer to nucleotide oligonucleotide polynucleotides or any fragments thereof. These terms also refer to DNA or RNA of genomic or synthetic origin, which can be single-stranded or double-stranded and can represent the sense or antisense strand of a peptide nucleic acid (PNA) or any DNA-like or RNA-like material. The term "operably linked" refers to a situation in which a first nucleic acid sequence amino acid sequence or part is in a functional relationship with a second nucleic acid sequence amino acid sequence or part. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. The dna sequence or protein or part that is operably linked can be in close proximity or contiguous, and when necessary, two protein coding regions are connected in the same reading frame.

[0050] The term "insertion" refers to a change in the amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.

[0051] The term "recombinant" refers to a polynucleotide of semisynthetic or synthetic origin that does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature. The term "recombinant" with respect to a viral vector refers to a vector, e.g., a viral genome, that has been manipulated in vitro, e.g., to express heterologous viral nucleic acid sequences using recombinant nucleic acid technology.

[0052] The term "chimeric" refers to a hybrid polynucleotide sequence formed by splicing polynucleotide sequences of different origins and functions through recombination. The term "chimeric classical swine fever virus" refers to a mutant classical swine fever virus whose genome has been modified through "recombination" and / or "chimerism" techniques.

[0053] Thus, in some embodiments, when the infectious polynucleotide is introduced into a cell, the infectious polynucleotide is transcribed, the viral genome is replicated, and infectious viral particles are produced.

[0054] In some embodiments, the infectious genomic cDNA further comprises vector sequences for replication in a prokaryotic host cell.

[0055] In some embodiments, the infectious genomic cDNA is present in a vector.

[0056] In some embodiments, the infectious polynucleotide is transcribed to produce a viral genome present in a viral particle.

[0057] In some embodiments, the infectious polynucleotide is a DNA and / or RNA polynucleotide.

[0058] In some embodiments, the infectious genomic plasmid is transcribed to produce a viral genome present in the cell.

[0059] Chimeric swine fever virus

[0060] On the other hand, the present invention discloses a chimeric classical swine fever virus particle. The classical swine fever virus particle has a microscopic regular dodecahedron envelope and a single-stranded RNA packaged in the envelope, the single-stranded RNA carrying a chimeric genome, the chimeric genome comprising an open reading frame and a 5'UTR region and a 3'UTR region located at both ends of the open reading frame. The nucleotide sequences of the 5'UTR region and the 3'UTR region are shown in SEQ ID NO.1-2, respectively. The development reading frame encodes Core, E rns (E0), E1, E2, N pro ,p7,NS2,NS3,NS4A,NS4B,NS5A and NS5B;Core,E rns (E0), E1, N pro The nucleotide sequences of the genes encoding the C-terminal region of the E2 protein, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B, and E2 protein are all derived from the genome of the C strain of the classical swine fever vaccine. The nucleotide sequence encoding the N-terminal antigenic region of the E2 protein is shown in SEQ ID NO. 3.

[0061] like Figure 1As shown, some embodiments provide a chimeric classical swine fever virus (rC / bUTRs-tE2). This virus was obtained by transfecting PK15 cells with the infectious clone pC / bUTRs-tE2 provided in the above embodiments and then rescuing the virus by passage in PK15 cells. Cellular characterization studies have shown that some embodiments of the chimeric classical swine fever virus (rC / bUTRs-tE2) also immunized rabbits with CSFV-specific and neutralizing antibodies 14 days after immunization.

[0062] In addition, some embodiments also immunized pigs with rC / bUTRs-tE2 and evaluated the vaccine safety, immunogenicity and protection against virulent infection of rC / bUTRs-tE2. The results showed that after the pigs were immunized with the chimeric swine fever virus rC / bUTRs-tE2, the pigs' rectal temperature was normal, there were no obvious clinical symptoms, the number of white blood cells and platelets were within the normal range, and no viremia or viral shedding was detected, indicating that rC / bUTRs-tE2 has good safety as a vaccine. 21 days after immunization, rC / bUTRs-tE2 can detect CSFV-specific antibodies and neutralizing antibodies in pigs. About 6-8 days after the infection, the pigs in the chimeric swine fever virus rC / bUTRs-tE2 immunization group had a slightly elevated body temperature but no obvious fever, and had mild clinical symptoms, but these symptoms recovered after 3-4 days. Seven days after infection, 10 2 The virus copy number was low, but 14 days after the infection, no virus was detected in the whole blood, throat swabs and anal swabs. After the infection, the titers of CSFV-specific antibodies and neutralizing antibodies in the rC / bUTRs-tE2-immunized pigs increased significantly. Until the end of the experiment, all pigs in the rC / bUTRs-tE2-immunized group survived well. 21 days after the infection, all pigs were euthanized and dissected, and no virus was detected in the tissues of the rC / bUTRs-tE2-immunized pigs. This shows that the chimeric swine fever virus rC / bUTRs-tE2 has good immunogenicity and can protect pigs against infection with virulent CSFV.

[0063] In addition, in order to evaluate the recognition potential of rC / bUTRs-tE2 as a marker vaccine, the indirect ELISA method based on BVDV tE2 serum antibodies disclosed in CN113512098A and the gene CSFV E2 established in the examples of this application were used. rns Serum antibody indirect ELISA was used to detect the serum of rabbits and pigs immunized with rC / bUTRs-tE2, and it was found that the serum of rabbits and pigs immunized with rC / bUTRs-tE2 showed CSFV E rns and BVDV E2 antibodies, but the sera of rabbits and pigs immunized with C strain showed CSFV Erns Antibody positive and BVDV E2 antibody negative. rns The double positivity of BVDVE2 and rC / bUTRs-tE2 antibodies can be considered as immunity to rC / bUTRs-tE2, while only CSFV E2 can be detected in the serum after wild-type CSFV infection. rns The chimeric CSFV rC / bUTRs-tE2 protein has the potential to serve as a marker vaccine and lays an important theoretical and technical foundation for the further development of chimeric marker vaccines for CSFV and BVDV E2 proteins.

[0064] Composition or labeled vaccine

[0065] Based on this, the present application also discloses a composition or vaccine. This composition or vaccine comprises chimeric classical swine fever virus particles and a pharmaceutically or veterinarily acceptable carrier, excipient, vehicle, or adjuvant. The chimeric classical swine fever virus particles are those provided in the above-mentioned embodiments, comprising a microscopic dodecahedral envelope and single-stranded RNA encapsulated within the envelope. This single-stranded RNA comprises a chimeric genome modified from the classical swine fever vaccine strain C. This chimeric genome has the same sequence as the infectious polynucleotide provided in the above-mentioned embodiments; the E2 protein in the envelope is the envelope protein provided in the above-mentioned embodiments.

[0066] The carrier, excipient, vehicle or adjuvant that can be used in combination with the vaccine of the present application is to be able to enhance the immune response of the pig to the composition or labeled vaccine. The carrier, excipient, vehicle or adjuvant can be used at the same time and the same position as the vaccine, or can be administered at different times, for example, as a booster. The carrier, excipient, vehicle or adjuvant can also be preferably administered to the pig in a manner and position different from that of administering the vaccine. Suitable carriers, excipients, vehicles or adjuvants are selected from aluminum hydroxide (alum), immunostimulating complexes (ISCOMS), nonionic block polymers or copolymers, cytokines (such as IL-1, IL-2, IL-7, IFN-α, IFN-β, IFN-γ, etc.), saponins, monophosphoryl lipid A (MLA), and muramyl dipeptide (MDP) etc. Other suitable adjuvants include, for example, potassium aluminum sulfate, heat-labile or heat-stable enterotoxin isolated from Escherichia coli, cholera toxin or its B subunit, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc. Toxin-based adjuvants such as diphtheria toxin, tetanus toxin and pertussis toxin can be inactivated before use, for example, by treating them with formaldehyde.

[0067] The new vaccines of the present application are not limited to any specific type or preparation method. The cloned viral vaccines include, but are not limited to, infectious DNA vaccines (i.e., DNA injected directly into pigs using plasmids, vectors or other conventional vectors), live vaccines, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, genetically engineered vaccines, etc. The vaccines are prepared by standard methods known in the art. The live viral vaccines are usually the most desirable vaccines, that is, they can activate all possible immune responses in the vaccine recipient, including systemic, local, humoral and cell-mediated immune responses. On the other hand, inactivated vaccines can only induce humoral immune responses. However, although live viral vaccines are the most ideal, they have several drawbacks, such as the potential risk of contamination by live accidental viral agents or the risk of the virus regaining virulence in the wild. It is worth noting that the examples of the present application provide a chimeric classical swine fever virus rC / bUTRs-tE2, which can produce specific antibodies and neutralizing antibodies in vivo, provide pigs with protection against virulent infection, and do not cause viremia and viral shedding in pigs, with good safety; the chimeric classical swine fever virus can also induce the production of specific BVDV E2 antibodies in the body, which can effectively distinguish vaccinated pigs from wild-type infected pigs, and has the application prospect of labeled vaccines.

[0068] Although live virus vaccines are most preferred, other types of vaccines can be used to vaccinate pigs with the new chimeric classical swine fever viruses disclosed herein. To prepare an inactivated virus vaccine, for example, virus propagation from an infectious cDNA clone is performed by methods known in the art or as disclosed herein. Serial virus inactivation is then optimized by methods generally known to those of ordinary skill in the art.

[0069] The term "immune response" refers to any response of the immune system of a subject (e.g., a human) to an antigen or antigenic determinant. Exemplary immune responses include humoral immune responses (e.g., the production of antigen-specific antibodies) and cell-mediated immune responses (e.g., the production of antigen-specific T cells). Assays for evaluating immune responses are known in the art and may include in vivo assays, such as assays for measuring antibody responses and delayed-type hypersensitivity reactions. In one embodiment, the analysis of antibody responses can primarily measure B cell function and B cell / T cell interactions. For antibody response assays, the antibody titers in the blood can be compared after antigen attack. "Antibody titer" as used herein can be defined as the highest dilution in serum after immunization, resulting in a value greater than the value of each subject's pre-immunization sample. In vitro assays may include assays for the ability of cell division or for providing help or releasing lymphokines and other expression activation and lysis target cell markers for other cell divisions. In one embodiment, lymphocytes from similar sources are compared, such as peripheral blood cells, spleen cells, or lymph node cells. However, as with the non-limiting examples of human peripheral blood cells and mouse spleen cells, it is possible to compare lymphocytes from different sources. For in vitro assays, cells (such as B cells, T cells and macrophages) can be purified or cells can be kept in their natural state (such as splenocytes or lymph node cells). Purification can be carried out by any method that provides the desired result. The proliferation capacity of cells can be tested in vitro with mitogens or specific antigens. In the presence of specific antigens, the ability of cell division can be measured using mixed lymphocyte reaction (MLR) assay. The supernatant from cultured cells can be tested for the ability to secrete specific lymphokines with quantitative cells. Cells can be removed from culture and tested for their ability to express activation antigens. This can be achieved by any suitable method, as in the non-limiting example of using antibodies or ligands in conjunction with the probes of the RNA encoding the activation antigens.

[0070] The term "marker vaccine" refers to a new generation of recombinant live vaccines that use genetic engineering technology to introduce molecular markers into the viral genome to distinguish it from wild strains.

[0071] Given that the current live attenuated swine fever vaccine strain C has a good protective effect, its replication efficiency is low, and the serotype detection after immunization cannot distinguish infected pigs from immune pigs. In the embodiment of the present application, the non-coding region and the main antigenic region of the E2 protein of the C strain are simultaneously replaced with the corresponding regions of BVDV to obtain a chimeric swine fever virus strain (named rC / bUTRs-tE2). The chimeric swine fever virus strain rC / bUTRs-tE2 can not only provide pigs with a protective effect against virulent infection, but also induce the production of CSFV E in rabbits and pigs. rnsand BVDV E2-specific antibodies, while wild-type CSFV infection can only induce the production of CSFV E rns Specific antibodies cannot induce BVDV E2-specific antibodies, so vaccinated pigs and wild-type classical swine fever virus-infected pigs can be distinguished serologically. The recombinant chimeric virus vaccine strain rC / bUTRs-tE2 has the recognition potential of a marker vaccine.

[0072] Construction of pC / bUTRs-tE2 infectious clone

[0073] For this reason, Figure 1 As shown, the present invention also provides a chimeric classical swine fever virus rC / bUTRs-tE2 of the classical swine fever virus strain C. This virus is obtained by replacing the non-coding region and the E2 major antigenic region of the attenuated live classical swine fever vaccine strain C with those derived from a BVDV strain. The non-coding region of the classical swine fever virus strain C is double-replaced with the BVDV non-coding region (5'UTR region and 3'UTR region) to obtain a chimeric classical swine fever virus cDNA infectious clone pC / bUTRs, and then the coding region of the major antigenic region domain I and domain II (aa 690-860) of E2 is replaced with the corresponding region of BVDV, thereby constructing the classical swine fever chimeric classical swine fever virus cDNA infectious clone pC / bUTRs-tE2.

[0074] In some embodiments, the pC / bUTRs-tE2 construction method uses overlap and enzyme ligation. Specifically, the construction process includes:

[0075] 1) Obtain BVDV 5'UTR fragment

[0076] BVDV 5'UTR was amplified using BVDV cDNA as template and specific primers b5'UTR-F / b5'UTR-R;

[0077] 2) Obtaining the infectious clone pPST1 / C of the swine fever vaccine strain C

[0078] Overlap PCR was then used to obtain the fusion fragment, and the BVDV 5'UTR was inserted into the swine fever vaccine C strain infectious clone pPST1 / C using the restriction endonuclease sites AatⅡ / Cla I. Specific reference was made to "Li, L., Pang, H., Wu, R., Zhang, Y., Tan, Y., Pan, Z., 2016. Development of a novel single-step reverse genetics system for the generation of classical swine fever virus. Arch Virol 161, 1831-1838".

[0079] 3) Obtain chimeric virus infectious clone pC / bUTRs

[0080] Based on this, the BVDV 3'UTR was amplified using BVDV cDNA as a template and specific primers b3'UTR-F / b3'UTR-R. Overlap PCR was then used to generate the fused fragment. The BVDV 3'UTR was then inserted into the swine fever vaccine C strain infectious clone pPST1 / C using the SpeⅠ / MluI restriction enzyme sites to generate the chimeric viral infectious clone pC / bUTRs. The full-length infectious clone pC / bUTRs with the substituted noncoding region was constructed according to the protocol described in "Pang, H.; Li, L.; Liu, H.; Pan, Z. Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis. Viruses 2021, 13, 1523."

[0081] 4) Obtaining the chimeric swine fever virus infectious clone pC / bUTRs-tE2

[0082] BVDV tE2 (aa690-860) was amplified using BVDV cDNA as a template and specific primers btE2-F / btE2-R, and then the fusion fragment was obtained by overlap PCR. BVDV tE2 was inserted into the infectious clone pC / bUTRs using the restriction endonuclease sites SpeⅠ / NcoⅠ to obtain the chimeric virus infectious clone pC / bUTRs-tE2 with the non-coding region and E2 replaced.

[0083] The structure of pC / bUTRs-tE2 is as follows Figure 2 As shown, the SDS-PAGE after double digestion of pC / bUTRs-tE2 with BamHI and MluI is shown in FIG. Figure 3 As shown, the band size is correct (the shaded segment in the figure is 5890 bp, and the remaining segments are 9208 bp), and the sequence is correct after sequencing, indicating that the chimeric swine fever virus infectious clone pC / bUTRs-tE2 with the 5'UTR region, 3'UTR region and E2 major antigen region of swine fever virus C strain replaced was successfully constructed.

[0084] The primers involved in this embodiment are shown in Table 1, where the underlined sequences are restriction endonuclease recognition sequences.

[0085] Table 1

[0086]

[0087] Rescue of chimeric swine fever virus rC / bUTRs-tE2

[0088] In some embodiments, the infectious cDNA clone pC / bUTRs-tE2 is transfected into PK15 cells using Lip3000, and pC / bUTRs (the full-length infectious clone of the chimeric virus replacing the non-coding region (5'UTR and 3'UTR) provided in the above embodiment) is transfected at the same time as a control. 72 hours after transfection, the cells are collected and immunofluorescence staining is performed using Anti-NS3 rabbit polyclonal antibody as the primary antibody for identification; the supernatant is collected to extract RNA, and RT-PCR is performed using specific primers to identify the replaced region and send the replaced region for sequencing. Furthermore, the supernatant is taken to infect PK15 cells again, and 48 hours later, the cells are collected and immunofluorescence staining is performed using Anti-NS3 rabbit polyclonal antibody as the primary antibody to identify the infectivity of the rescued classical swine fever virus strain C non-coding region and the E2-replaced chimeric classical swine fever virus. The results are shown in FIG. Figure 4 As shown in the figure, the immunofluorescence results of the cells after transfection showed that the NS3 protein of rC / bUTRs and rC / bUTRs-tE2 was expressed in the cells, indicating that the infectious cDNA clone plasmid was correctly expressed in the cells after transfection. However, from the preliminary analysis of the number of positive cells, the positive cells of rC / bUTRs-tE2 were significantly less than those of rC / bUTRs. The RT-PCR results of the supernatant after transfection are shown in the figure. Figure 5 As shown, rC / bUTRs and rC / bUTRs-tE2 were both positive in BVDV 5'UTR and 3'UTR detection, rC / bUTRs was negative in BVDV E2 detection, while rC / bUTRs-tE2 was positive. The size of the target band was consistent with the expected result, which preliminarily proved that chimeric swine fever virus particles were released in the rescued supernatant. The subsequent RT-PCR target band sequencing results proved that the genome of the supernatant virus was correctly replaced.

[0089] Cell passaging of rC / bUTRs-tE2

[0090] In some embodiments, the transfected cells were passaged, and representative passage infected cells were frozen and thawed three times, the supernatant was collected by centrifugation and infected with PK15 cells. After 48 hours, the cells were collected and immunofluorescence staining was performed using Anti-NS3 rabbit polyclonal antibody as the primary antibody to identify the infectivity of the passaged CSFV C strain noncoding region and E2-substituted chimeric CSFV. The transfected PK15 cells were named rC / bUTRs-tE2 P0, P10, P20, P30 and P40. The results are as follows: Figure 6 and7 As shown in the figure, starting from rC / bUTRs-tE2 P20, the infectivity of rC / bUTRs-tE2 on PK15 cells gradually increased and stabilized with cell passage. The viral titer of rC / bUTRs-tE2 on K15 cells increased significantly and stabilized starting from rC / bUTRs-tE2 P20. This indicates that the infectivity of rC / bUTRs-tE2 increased and stabilized after the 20th passage.

[0091] Genetic stability of rC / bUTRs-tE2

[0092] Table 2 Nucleotide and amino acid mutations of rC / bUTRs-tE2 at passages 0, 30, 40, and 60

[0093]

[0094] The whole genomes of rC / bUTRs-tE2 P0, P30, and P40 were sequenced, and the sequencing results are shown in Table 2. In Table 2, "a" represents the nucleotide position, and the first nucleotide position of the 5'UTR is defined as 1; "b" represents the amino acid position, and the first amino acid position of the polyprotein is defined as 1.

[0095] Compared to the original genome P0, rC / bUTRs-tE2 P30 exhibited two nucleotide mutations in the E2 gene and one nucleotide mutation in NS5B. Correspondingly, two amino acid mutations, M834K and M979K, occurred in the E2 protein. However, the NS5B mutations were synonymous, and the nucleotide mutations did not result in amino acid changes. Compared to the 30th generation genome, the 40th generation rC / bUTRs-tE2 genome exhibited both nucleotide and amino acid changes at the mutation sites. Genome sequencing results showed that, consistent with viral passage results, the chimeric CSFV rC / bUTRs-tE2 P30 underwent significant changes in properties, similarly altering the genome. rC / bUTRs-tE2 P40 showed no significant differences in viral properties compared to P30, and similarly, the genome remained unchanged. These results indicate that rC / bUTRs-tE2 P30 is relatively genetically stable in PK15 cells.

[0096] Safety of rC / bUTRs-tE2D virus as a marker vaccine

[0097] This study investigated the cell tropism, growth curve, genome replication efficiency, and plaque formation ability of the chimeric classical swine fever virus rC / bUTRs-tE2 (hereinafter referred to as the 30th generation and subsequent generations).

[0098] In one test case, rC / bUTRs-tE2 was used to infect PK15 cells and MDBK cells, respectively, and CSFV C strain and chimeric CSFV rC / bUTRs were used as controls. 48 hours after infection, cells were indirect immunofluorescence stained using Anti-NS3 rabbit polyclonal antibody as the primary antibody. The results are shown in Figure 2. Figure 8 As shown, rC / bUTRs-tE2 and rC / bUTRs retained the ability of classical swine fever virus C strain to infect PK15 cells, but the infectivity of rC / bUTRs-tE2 to PK15 cells was significantly reduced compared with rC / bUTRs and classical swine fever virus C strain; classical swine fever virus C strain, rC / bUTRs-tE2 and rC / bUTRs all showed extremely low infectivity to MDBK cells.

[0099] In one test case, PK15 cells were infected with swine fever virus C strain, rC / bUTRs, and rC / bUTRs-tE2 at an MOI of 0.001. The cells were harvested 6 and 12 hours after infection, total RNA was extracted, and the viral genome copy number was determined by RT-qPCR. The results were as follows: Figure 10 As shown in the figure, the genome copy number of rC / bUTRs at 6h and 12h was significantly higher than that of strain C, which indicates that non-coding region replacement can improve the replication efficiency of classical swine fever virus strain C; while the genome copy number of rC / bUTRs-tE2 at 6h and 12h was significantly lower than that of strain C, indicating that the replacement of E2 significantly downregulated the replication of the virus in PK15 cells.

[0100] In one test case, swine fever virus C strain, rC / bUTRs and rC / bUTRs-tE2 were each 50 SK6 cells were inoculated with the virus and incubated for 1 hour, then replaced with 1.5% carboxymethyl cellulose for 96 hours, and the plaque-forming ability of the mutant virus was determined by color development. Figure 11 As shown, ImageJ software analysis revealed that the size of plaques formed by rC / bUTRs was not significantly different from that of strain C, while the plaques formed by chimeric CSFV rC / bUTRs-tE2 were smaller than those of strain C. These results indicate that substitutions in the noncoding regions have no effect on the plaque-forming ability of CSFV strain C, that is, do not affect the rate of viral transmission between cells; substitutions in E2 significantly downregulated the virus's plaque-forming ability and significantly reduced the rate of viral transmission between cells.

[0101] These results indicate that the replacement of non-coding regions and E2 has no obvious effect on the cell tropism of CSFV C strain, indicating that the host range of chimeric CSFV rC / bUTRs-tE2 will not expand and is safe.

[0102] Immunogenicity of rC / bUTRs-tE2 in rabbits

[0103] Classical Chinese cholera virus strain C is a lapinized strain obtained by multiple passages of the virulent strain Shimenzhu in rabbits. Classical Chinese cholera virus strain C can replicate in the rabbit spleen, induce rabbit fever, and elicit an antibody immune response. Therefore, rabbits are an ideal organism for evaluating live attenuated Classical Chinese cholera vaccines. This example further investigates the effects of substitutions in the noncoding region and E2 on the viral properties and immunogenicity of Classical Chinese cholera virus strain C in rabbits.

[0104] In one test case, rabbits were immunized with CSFV strain C, rC / bUTRs, and rC / bUTRs-tE2, respectively. Rectal temperatures were measured every 6 hours from 0 to 72 hours after immunization. Three days after immunization, half of the rabbits (three rabbits) in each group were randomly euthanized, and spleen tissue was obtained for viral copy number determination. Serum was collected 7 and 14 days after immunization to determine serum CSFV E2-specific antibody and virus neutralization titers. The results, as shown in Table 3, showed that neither rC / bUTRs nor rC / bUTRs-tE2 induced rabbit fever but could replicate in the rabbit spleen. In contrast, strain C induced both typical rabbit fever and replication in the rabbit spleen.

[0105] Table 3 Rabbit experiments on strain C, rC / bUTRs and rC / bUTRs-tE2

[0106]

[0107] like Figure 12 As shown in the left figure, on the 7th day after immunization, the serum of rabbits immunized with strain C and rC / bUTRs tested positive for CSFV-specific antibodies; on the 14th day after immunization, the serum of rabbits immunized with rC / bUTRs-tE2 tested positive for CSFV-specific antibodies; and on the 7th and 14th days after immunization, the levels of CSFV-specific antibodies induced by C / bUTRs-tE2 were significantly lower than those of strain C and rC / bUTRs.

[0108] like Figure 12 As shown in the right figure, on day 14 after immunization, the virus neutralizing antibody titer induced by rC / bUTRs-tE2 was 1:16-1:32, while the virus neutralizing antibody titer induced by the CSFV vaccine strain C and the recombinant chimeric virus vaccine strain rC / bUTRs was 1:32-1:64. The serum virus neutralizing antibody titer and serum CSFV E2-specific antibody results were similar. The antibody immunity induced by rC / bUTRs-tE2 in rabbits was significantly lower than that induced by the CSFV vaccine strain C and the recombinant chimeric virus vaccine strain rC / bUTRs, indicating that although the E2 replacement resulted in a weakened specific antibody immune response induced by the chimeric CSFV rC / bUTRs-tE2 in rabbits, it could still induce CSFV E2-specific antibodies and neutralizing antibodies in rabbits.

[0109] Immunogenicity and protective effect of rC / bUTRs-tE2 in pigs

[0110] In one test case, 11 5-week-old piglets, negative for both antibodies and antigens to classical swine fever virus, were randomly divided into 3 groups, 3 of which were immunized with classical swine fever virus strain C, 5 were immunized with rC / bUTRs-tE2, and 3 were immunized with DMEM culture medium. They were acclimated for one week before the start of the experiment. The pigs were immunized with classical swine fever vaccine strain C by intramuscular injection at a dose of 10 4 TCID 50 / (1mL), the recombinant chimeric virus vaccine strain rC / bUTRs-tE2 was administered by intramuscular injection at a dose of 10 4.5 TCID 50 / pig (1mL), DMEM was injected intramuscularly, the inoculation dose was 1mL / pig, and the animals were observed for 30 minutes after inoculation; all pigs were boosted with the same dose and the same method 14 days after immunization. Pigs were challenged with CSFV virulent Shimen strain by intranasal drops 28 days after immunization, with a challenge dose of 10 5.5 TCID 50 / (1 mL), observe the animals for 30 minutes after the infection. Rectal temperature of pigs: starting from day 0 of immunization, monitor and record the rectal temperature of all pigs every day. When the rectal temperature of the pig is ≥40.5℃, it is considered to have a fever. Scoring of clinical symptoms of classical swine fever: starting from day 0 of immunization, all pigs are scored according to the classical swine fever clinical scoring standard established by Mittelholzer et al., including vitality, body tension, body shape, breathing, walking, skin (especially ears, nose, legs and tail), eyes / conjunctiva, appetite, defecation and residual feed. Normal clinical symptoms are 0 points, mild clinical symptoms are 1 point, obvious clinical symptoms are 2 points, severe clinical symptoms are 3 points, all scores are added up, and the maximum clinical symptom score is 30 points. Pig blood was collected through the anterior vena cava at 0, 7, 14, 21, and 28 days after immunization and 0, 3, 7, 14, and 21 days after infection. Part of the blood was packaged in EDTA anticoagulant tubes for subsequent detection of viremia and white blood cell and platelet counts. Viremia was detected by extracting whole blood RNA and then determining the number of viral copies in the blood by RT-qPCR. The white blood cell and platelet counts were determined using a SYSMEX blood cell analyzer at Wuhan Children's Hospital. The other part was used to separate pig serum. The collected pig blood was allowed to stand at 37°C for 1 hour, then at 4°C overnight, and then centrifuged at 4°C and 5000 rpm for 20 minutes. The serum was collected, packaged, and stored at -80°C for later use. Throat and anal swabs were collected with cotton swabs at 0, 7, 14, 21, and 28 days after immunization and 0, 3, 7, 14, and 21 days after challenge. The swabs were dissolved in 1 mL of PBS (with 2% antibiotics) and total RNA was extracted. The viral copy number in the swabs was measured by RT-qPCR to detect viral shedding. Pigs that were moribund after challenge and those that survived 21 days after challenge were euthanized and dissected. Tonsils, mandibular lymph nodes, kidneys, and spleens were obtained. Total RNA was extracted and the viral copy number in the tissues was measured by RT-qPCR to detect the viral content in the pig tissues.

[0111] The results are as follows Figure 13 As shown in the figure, pigs in the chimeric swine fever virus rC / bUTRs-tE2 immunization group had a slight increase in rectal temperature around 7 days after infection, but no obvious fever was observed, below 40.5℃, and returned to normal after 2-3 days until the end of the experiment. Figure 14 As shown in the left figure, pigs in the chimeric swine fever virus rC / bUTRs-tE2 immunization group developed mild clinical symptoms around 7 days after infection, including mild diarrhea, loss of appetite, and physical exhaustion leading to lying down. However, these mild clinical symptoms recovered after 2-3 days, and the pigs survived well until the end of the experiment ( Figure 14 (right image). Figure 15 As shown, the number of white blood cells and platelets in the pigs immunized with chimeric swine fever virus rC / bUTRs-tE2 decreased slightly 7 days after infection, but returned to normal 14 days after infection until the end of the experiment.

[0112] Table 4 shows the results of RT-qPCR detection of viral RNA copy numbers in pig whole blood, throat swabs and anal swabs, where "-" indicates no detection, " / " indicates death, and the number in brackets on the day after immunization is the number of immunized pigs that died or were detected with viral RNA.

[0113] Table 4 RT-qPCR detection of viral RNA copy numbers in pig whole blood, throat swabs and anal swabs

[0114]

[0115]

[0116] As shown in Table 4, (9.27±9.95)×10 2 Very low copy number of viral RNA was detected in the whole blood 14 days after the challenge, that is, mild viremia occurred 7 days after the challenge, and then returned to normal 14 days after the challenge. No viral RNA was detected in the anal swabs of pigs in the chimeric swine fever virus rC / bUTRs-tE2 immunization group from the challenge to the end of the experiment, but (6.90±10.2)×10 2 Extremely low copy numbers of viral RNA were detected, followed by no viral RNA being detected in throat swabs 14 days after challenge. Lower levels of virus shedding were detected in throat swabs, but returned to normal after 14 days.

[0117] Table 5 shows the viral RNA content in pig tissues (tonsils, submandibular lymph nodes, spleen, and kidneys) detected by RT-qPCR. In Table 5, "-" indicates no detection. As shown in Table 5, no viral RNA was detected in any tissue of pigs in the chimeric swine fever virus rC / bUTRs-tE2 immunization group. These results indicate that the recombinant chimeric virus vaccine strain rC / bUTRs-tE2 can induce protection in pigs against virulent lethality.

[0118] Table 5 Detection of viral RNA content in pig tissues by RT-qPCR

[0119] Grouping tonsil submandibular lymph nodes spleen kidney C strain - - - - rC / bUTRs-tE2 - - - - DMEM <![CDATA[(6.81±8.74)×10 6 ]]> <![CDATA[(9.37±4.55)×10 6 ]]> <![CDATA[(5.15±3.71)×10 5 ]]> <![CDATA[(5.59±3.52)×10 4 ]]>

[0120] like Figure 16As shown in the left figure, 21 days after immunization, pigs in the rC / bUTRs-tE2-immunized group were positive for CSFV antibodies. At 28 days after immunization, CSFV antibody blocking values in the rC / bUTRs-tE2-immunized group ranged from 48.44% to 63.37% (a blocking value ≥ 40% was considered antibody positive). CSFV antibody levels in the rC / bUTRs-tE2-immunized group increased significantly after challenge. At 21 days after challenge, the rC / bUTRs-tE2-immunized group had an average blocking rate of 88.17% for CSFV-specific antibodies. Immunization of pigs with the chimeric swine fever virus rC / bUTRs-tE2 induced the production of CSFV-specific and virus-neutralizing antibodies.

[0121] like Figure 16 As shown in the figure on the right, the virus neutralization antibody titer in the serum was determined by virus neutralization experiment. Generally, a neutralization antibody titer of ≥1:16 is considered positive. It was found that 14 days after immunization, the virus neutralization antibody titer of the rC / bUTRs-tE2 immunized group was very low, with an antibody titer of ≤1:16; 28 days after immunization, the virus neutralization antibody titer of the rC / bUTRs-tE2 immunized group was 1:16-1:64. After the virus challenge, the neutralization antibody titer of the rC / bUTRs-tE2 immunized group increased significantly. 21 days after the virus challenge, the neutralization antibody titer of the rC / bUTRs-tE2 immunized group reached 1:256-1:1024.

[0122] Indirect ELISA based on rC / bUTRs-tE2

[0123] Since the marker vaccine can distinguish vaccinated pigs from wild infected pigs while having a good protective effect, the marker vaccine is a very promising research direction for the current swine fever vaccine. According to previous research findings, the chimeric swine fever virus rC / bUTRs-tE2 can be used as a potential candidate marker vaccine for swine fever virus. In order to evaluate the potential role of the chimeric swine fever virus rC / bUTRs-tE2 as a marker vaccine, the present embodiment also uses a specific CSFV E rns and BVDVE2 proteins as coating antigens, two indirect ELISA methods for serum antibody detection were established to distinguish vaccinated pigs from wild infected pigs.

[0124] In one embodiment, based on CSFV E rns The indirect ELISA method using protein as coating antigen was established by referring to the method disclosed in CN113512098A to detect rabbit serum and pig serum immunized with rC / bUTRs-tE2. rns The antigen (refer to CN113512098A) can be obtained by constructing a recombinant expression vector, recombinantly expressing in Escherichia coli and purifying it.rns The indirect ELISA method was used to detect rabbit serum and pig serum immunized with rC / bUTRs-tE2. Figure 17 Left: 14 days after immunization, the rabbit serum of swine fever vaccine C strain, rC / bUTRs and rC / bUTRs-tE2 immunization groups all showed CSFV E rns Antibody positive; Figure 17 Right: 21 days after immunization, the pig serum of the swine fever vaccine C strain and the rC / bUTRs-tE2 immunization group showed CSFV E rns Antibody positive; CSFV E was detected in both rabbit and pig serum in the DMEM immunization group. rns Antibody.

[0125] In one embodiment, an indirect ELISA method based on BVDV tE2 protein as the coating antigen was established with reference to the method disclosed in CN113512098A to detect rabbit serum and pig serum immunized with rC / bUTRs-tE2. The results are shown in FIG. Figure 18 As shown, it was found that 14 days after immunization, the rabbit serum of the rC / bUTRs-tE2 immunized group showed positive BVDV E2 antibodies, but the rabbit serum of the C strain and DMEM immunized group was negative for BVDV E2 antibodies. 21 days after immunization, the pig serum of the rC / bUTRs-tE2 immunized group began to show positive BVDV E2 antibodies, but the pig serum of the C strain and DMEM immunized group was negative for BVDV E2 antibodies. The results showed that the recombinant chimeric virus vaccine strain rC / bUTRs-tE2 can induce the production of BVDV E2 antibodies in rabbits and pigs that are different from those infected with wild-type swine fever virus, and can be identified by the BVDV tE2 indirect ELISA method, indicating that rC / bUTRs-tE2 has the ability to be used as a marker vaccine. When CSFV E rns When the chimeric rC / bUTRs-tE2 antibody is double positive, it can be considered as immunity, while wild-type CSFV infection can only induce the production of CSFV E2. rns Specific antibodies cannot induce the production of BVDV E2-specific antibodies, thereby achieving the purpose of distinguishing vaccinated pigs from wild-type swine fever virus-infected pigs.

[0126] CSFV E rns Recombinant expression and purification

[0127] In a CSFV E rns In the construction and identification of expression vectors, specific primers E rns -F (as shown in SEQ ID NO.4) and E rns-R, using the synthetic gene (as shown in SEQ ID NO.5) as a template for PCR amplification of CSFV E rns The target gene was cloned and the NcoⅠ and XhoⅠ restriction sites were introduced at both ends. rns The target gene was inserted into the pET-28a expression vector, which contains a 6×His tag at the end of the inserted gene. DH10B or DH5α cloning competent cells were then transformed. Positive clones were screened by colony PCR. The extracted plasmid was digested with NcoⅠ and KpnⅠ for identification. The results were as follows: Figure 19 The plasmid identified by enzyme digestion was selected and sent for sequencing to construct the correct CSFV E rns The expression vector was named pET-28a-E rns .

[0128] In a CSFV E rns In the example of protein induction expression and identification, pET-28a-E rns Transform expression competent cells E. coli BL21, pick a single clone, and culture in LB medium containing kanamycin at 37℃ with shaking. 600 When the value reaches 0.6-0.8, IPTG is added to a final concentration of 1mM and the induction culture is continued at 37℃ for 4h. The cells are collected and ultrasonically disrupted. The whole cells, lysis supernatant and cell pellet are analyzed by SDS-PAGE. The results are as follows Figure 19 As shown in the left figure. The expression of CSFV E was analyzed by software rns The protein size is about 26.7 kDa. rns The target protein was successfully induced to express, with a size between 25kDa and 35kDa, which was consistent with the expectation. And from the analysis of the bands, CSFV E rns The target protein is expressed in the bacterial precipitate. rns The target protein was fused with a 6×His tag at the C-terminus and Western-Blot analysis was performed using Anti-His as the primary antibody. The results were as follows: Figure 19 As shown in the right figure, the target band of Western blot is consistent with the target band of SDS-PAGE, indicating that CSFV E rns The target protein is expressed correctly.

[0129] In a CSFV E rns In the recombinant protein expression and purification example, the BL21 / pET-28a-E rns The bacterial solution was inoculated into 1 L of LB medium containing kanamycin at a volume ratio of 1:100 and cultured in a 37°C bacterial incubator with shaking.600 When the value reaches 0.6-0.8, IPTG is added to a final concentration of 1mM and the induction culture is continued at 37°C for 4h. After the induction culture is completed, the supernatant is discarded by centrifugation and the precipitate is collected. The precipitate is resuspended in lysis buffer, ultrasonically disrupted at low temperature, and then centrifuged and the precipitate is collected and purified by Ni-His nickel column affinity chromatography to obtain the target protein CSFV E rns The purified eluate was subjected to SDS-PAGE analysis, and the results were as follows: Figure 20 Lane 1 is the supernatant after binding, which contains only a small amount of target protein, indicating that most of the target protein is bound to the nickel column; lanes 2-4 are the beginning, middle, and end of the miscellaneous protein eluate, respectively, showing that only a small amount of target protein is eluted; lanes 5-14 are the target protein eluate, showing a single and large protein band between 25-35 kDa, which is consistent with the expected size, that is, the purified target protein CSFV E rns .

[0130] The collected target protein eluate was then dialyzed for refolding. A gradient dilution refolding solution was used at 4°C to remove imidazole and urea from the protein. The dialyzed refolded target protein CSFV E rns SDS-PAGE analysis was performed, and the results were as follows Figure 21 Lane 5 is the target protein CSFV E after dialysis and refolding. rns The band was single, high purity, and the size was between 25-35 kDa, which was consistent with the expectation. rns The protein concentration was determined and the aliquots were stored at -80°C for later use.

[0131] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. An isolated infectious polynucleotide or infectious clone, wherein the infectious polynucleotide or the infectious clone carries a chimeric genome, wherein the chimeric genome comprises an open reading frame and a 5'UTR region and a 3'UTR region located at both ends of the open reading frame; The 5'UTR region and the 3'UTR region are derived from BVDV, and their nucleotide sequences are shown in SEQ ID NO. 1-2 respectively; The open reading frame encodes Core, E rns (E0), E1, E2, N pro ,p7,NS2,NS3,NS4A,NS4B,NS5A and NS5B;Core,E rns (E0), E1, N pro The nucleotide sequences of the genes encoding the C-terminal region of p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B, and E2 were all derived from the genome of the C strain of classical swine fever vaccine; The N-terminal antigenic region of the E2 protein is derived from BVDV, and its encoding nucleotide sequence is shown in SEQ ID NO.3; Introducing the infectious polynucleotide or the infectious clone into cells and producing infectious classical swine fever virus particles through passaging; The infectious polynucleotide or infectious clone further comprises a vector sequence that replicates in a prokaryotic host cell; the infectious polynucleotide or infectious clone is a DNA polynucleotide or an RNA polynucleotide; The infectious polynucleotide or infectious clone is used to protect pigs against virulent infection of classical swine fever virus; the infectious polynucleotide or infectious clone induces the production of distinguishable BVDV E2-specific antibodies.

2. A vector comprising the infectious polynucleotide or infectious clone according to claim 1.

3. A cell carrying the infectious polynucleotide or infectious clone according to claim 1.

4. A classical swine fever virus particle, wherein the classical swine fever virus particle encapsulates a single-stranded RNA carrying a chimeric genome, wherein the chimeric genome comprises an open reading frame and a 5'UTR region and a 3'UTR region at both ends of the open reading frame; The nucleotide sequences of the 5'UTR region and the 3'UTR region are shown in SEQ ID NO. 1-2, respectively; The open reading frame encodes Core, E rns (E0), E1, E2, N pro ,p7,NS2,NS3,NS4A,NS4B,NS5A and NS5B;Core,E rns (E0), E1, N pro The nucleotide sequences of the genes encoding the C-terminal region of p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B, and E2 were all derived from the genome of the C strain of classical swine fever vaccine; The nucleotide sequence encoding the N-terminal antigen region of the E2 protein is shown in SEQ ID NO.

3.

5. A composition or a labeled vaccine, comprising classical swine fever virus particles encapsulating a single-stranded RNA carrying a chimeric genome, wherein the chimeric genome comprises an open reading frame and 5'UTR and 3'UTR regions at both ends of the open reading frame; The nucleotide sequences of the 5'UTR region and the 3'UTR region are shown in SEQ ID NO. 1-2, respectively; The open reading frame encodes Core, E rns (E0), E1, E2, N pro ,p7,NS2,NS3,NS4A,NS4B,NS5A and NS5B;Core,E rns (E0), E1, N pro The nucleotide sequences of the genes encoding the C-terminal region of p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B, and E2 were all derived from the genome of the C strain of classical swine fever vaccine; The nucleotide sequence encoding the N-terminal antigen region of the E2 protein is shown in SEQ ID NO.

3.

6. The composition or labeled vaccine according to claim 5, further comprising a pharmaceutically or veterinarily acceptable excipient; Optionally, the excipient is selected from aluminum hydroxide, immunostimulatory complexes, nonionic block polymers, cytokines, saponins, monophosphoryl lipid A, and muramyl dipeptide, potassium aluminum sulfate, heat-labile or heat-stable enterotoxin isolated from Escherichia coli, cholera toxin or its B subunit, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant.

7. The composition or labeled vaccine according to claim 5, which is an attenuated vaccine.

8. Use of the infectious polynucleotide or infectious clone according to claim 1 in the preparation of a swine fever marker vaccine.

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