A reverse genetics operating system for genome segment cloning and rescue of the pseudorabies virus Bartha K61 vaccine strain, its construction method and application

Abstract

This invention relates to a reverse genetics operating system for the segmented cloning and rescue of the Bartha K61 pseudorabies virus vaccine strain genome, its construction method, and its application. The system includes: recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG containing the complete genome of the Bartha K61 pseudorabies virus vaccine strain, and host cells expressing the complete genome of the Bartha K61 pseudorabies virus vaccine strain. The reverse genetics operating system for the Bartha K61 pseudorabies virus vaccine strain of this invention successfully segments the Bartha K61 genome into six segments using ExoCET cloning and assembly technology, and then assembles them into three recombinant plasmids: BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG. These three recombinant plasmids, after PacI linearization, can be co-transfected into African green monkey kidney cells to rescue the Bartha K61 virus, and the infectivity of the rescued Bartha K61 virus is comparable to that of the vaccine strain. Therefore, this reverse genetics operating system can provide an excellent vector for creating live vector vaccines related to the prevention and treatment of swine diseases, and can be used to develop Bartha K61 recombinant genetic engineering vector vaccines that express relevant swine disease antigen genes.

Description

Technical Field

[0001] This invention relates to a reverse genetics operating system for segmented cloning and rescue of the pseudorabies virus Bartha K61 vaccine strain genome, its construction method and application, belonging to the field of molecular biology technology. Background Technology

[0002] Pseudorabies virus (PRV) is a member of the alpha-herpesvirus subfamily of the herpesviridae family. It is the pathogen of pseudorabies, causing symptoms in the respiratory, nervous, and reproductive systems of the host. Pseudorabies causes severe economic losses to livestock farms worldwide. The pseudorabies virus genome is a double-stranded DNA, 150kb in length, containing a long unique region (UL), a short unique region (US), and intermediate repeat sequences (IRS) and terminal repeat sequences (TRS) at both ends of the US region. Bartha K61 is one of the classic naturally attenuated live virus vaccines for the prevention and treatment of pseudorabies. It was obtained by Hungarian virologist Adorján Bartha through continuous passage in cells using a virulent strain of porcine pseudorabies virus as a parent, and is widely used in pig farms worldwide.

[0003] Since the discovery of the attenuated Bartha K61 strain, researchers have not only explored its immunoprotective efficacy as a PRV vaccine strain, but also preliminarily elucidated its attenuation mechanism compared to the wild-type strain at the cellular and animal levels. They confirmed that complete deletions of the US8 (gE) and US9 genes, as well as partial deletions of US7 (gI) and US2 genes in the short unique region (US), are crucial for attenuating Bartha virulence. This provides important reference for subsequent genetic engineering production of attenuated live vaccines. gE and gI glycoproteins can form heterodimers and jointly participate in viral virulence, which is a necessary condition for the anterograde transport of viral particles in neurons. US9 also plays a role in anterograde propagation in neurons; therefore, deletion of the US9 gene disrupts the anterograde neuronal transport pathway of Bartha K61.

[0004] BarthaK61 has many advantages as a viral vector: (1) High safety: The Bartha K61 vaccine strain was successfully developed in 1961 and has been widely used worldwide for more than 60 years. It is safe and effective, and some countries have eradicated swine pseudorabies as a result. (2) Large capacity for carrying foreign genes: The Bartha K61 genome is 137kb long, with non-essential genes accounting for about 40%, and the theoretical maximum capacity can reach 14kb. (3) Wide host range: Livestock such as cattle, pigs, horses, cats, and dogs, as well as some carnivorous wild animals, can be vaccinated with the Bartha K61 vaccine strain. (4) It has been successfully used as a viral vector to develop a variety of vaccines. (5) Long duration of immunity. These advantages have led researchers to use it to produce viral vector vaccines, which can be used not only to prevent pseudorabies virus itself, but also to prevent other viruses, such as porcine reproductive and respiratory syndrome virus and swine influenza virus. Moreover, BarthaK61 has been used as a vector platform for loading other distantly related PRV strains and other pathogen antigens. For example, to effectively combat the recent emergence of PRV strains in China, researchers loaded viral antigens with immunogenicity to variant strains (such as gB, gD, and gC) into the Bartha K61 vector, improving the animal protective efficacy of the Bartha K61 vaccine against PRV variants. Introducing the GP5 gene of porcine reproductive and respiratory syndrome virus, the hemagglutinin gene of swine influenza H3N2, and the HA hemagglutinin or N1 neuraminidase gene of swine influenza H1N1 into the Bartha K61 vector showed significant clinical protection after inoculation of domestic pigs.

[0005] Red / ET is a gene editing technology that precisely modifies DNA molecules by utilizing homologous recombination between circular and linear DNA fragments mediated by Redα / Redβ recombinases, or homologous recombination between linear DNA fragments mediated by RecE / RecT recombinases. Redα and RecE are 5'→3' exonucleases that digest double-stranded DNA to form a 3' single-stranded DNA (ssDNA), exposing the single-stranded ends. Redβ and RecT are ssDNA-binding proteins that bind to the digested ssDNA, protecting it from nuclease degradation and mediating homologous recombination between the ssDNA fragment and the target DNA sequence. Compared to traditional genetic manipulation, Red / ET recombination engineering is not limited by exonuclease cleavage targets, does not require the artificial introduction of DNA ligases, is not limited by restriction enzyme sites or DNA size, and requires shorter homologous sequences, making it particularly suitable for genetic manipulation of DNA molecules larger than 10kb.

[0006] Based on this, Wang et al. combined RecET-mediated in vivo homologous recombination with exonuclease-mediated in vitro recombination to develop ExoCET technology, which can efficiently, rapidly and accurately achieve the targeted capture of genome fragments >100kb and the assembly of >13 gene fragments in E. coli (Wang, H., et al., ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. Nucleic Acids Research, 2018, 46(5), e28.). Zhang et al. cloned the adenovirus genome into the p15A vector using RecE / RecT recombination, and then modified the adenovirus genome through Redα / Redβ-mediated linear circular recombination, thus designing a complete step for the engineering cloning and modification of adenovirus technology (Zhang, W., et al., An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity. 2017.19(8):p.1698-1709). Based on this, Liu et al. constructed a recombinant adenovirus vaccine expressing the VP2 protein of infectious bursal disease virus (IBDV) (Liu Ruxin, Construction of recombinant avian adenovirus expressing the VP2 protein of IBDV, Shandong University).

[0007] Reverse genetics refers to the study of the heredity and variation of organisms from the perspective of the biological genome and its contained biological information, using a gene-to-trait research approach. The core of viral reverse genetics is the construction of infectious DNA molecular clones, which, through in vitro or in vivo transcription, rescue live viruses with similar biological characteristics to the parent strain. This allows for research into the structure and function of the viral genome, pathogenic mechanisms, and molecular immune mechanisms. However, for the 137kb-long pseudorabies virus Bartha K61 vaccine strain, not only is it enormous, but it also contains numerous internal and terminal repetitive sequences, making direct cloning of the complete genome extremely difficult. Even when using segmented cloning and reassembling to form a complete genome, the assembly difficulty increases with the length of the genome fragments. Therefore, establishing a reverse genetics system for segmented cloning and rescue of the large Bartha K61 genome would greatly facilitate genome cloning and modification, significantly promoting and advancing basic research related to Bartha K61. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a reverse genetics operating system for genome segmentation cloning and rescue of the pseudorabies virus Bartha K61 vaccine strain, along with its construction method and application.

[0009] Terminology Explanation:

[0010] Redαβ linear-circular recombination: λ phage recombinant proteins Redα and Redβ can efficiently mediate homologous recombination between linear and circular DNA in *E. coli* cells. Redα is a 5'-3' exonuclease, and Redβ is a single-stranded DNA annealing protein. (Wang H, Li Z, Jia R, Hou Y, Yin J, Bian X, et al. RecET direct cloning and Redabrecombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. *Nature Protocols*. 2016; 11(7):1175-90.)

[0011] RecET linear recombination: Rac prophage recombinant proteins RecE and RecT can efficiently mediate homologous recombination between linear DNAs in E. coli cells. RecE is a 5'-3' exonuclease, and RecT is a single-stranded DNA annealing protein. (Wang H, Li Z, Jia R, Hou Y, Yin J, Bian X, et al. RecET direct cloning and redabrecombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nature Protocols. 2016; 11(7):1175-90.)

[0012] ExoCET cloning technology: a gene cloning method combining exonuclease-mediated in vitro homologous recombination and RecET linear recombination. (Wang H, Li Z, Jia R, Yin J, Li A, Xia L, et al. ExoCET: exonuclease invitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. Nucleic Acids Res. 2018; 46(5):e28.)

[0013] The technical solution of the present invention is as follows:

[0014] A reverse genetics operating system for segmented cloning and rescue of the genome of the pseudorabies virus Bartha K61 vaccine strain, comprising: recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG containing the complete genome of the pseudorabies virus Bartha K61 vaccine strain, and host cells expressing the complete genome of the pseudorabies virus Bartha K61 vaccine strain.

[0015] According to a preferred embodiment of the present invention, the construction method of the recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG is as follows:

[0016] (1) The Bartha K61 genome was cut into six segments: A segment 26kb, B segment 12kb, C segment 30kb, F segment 32kb, G segment 19kb and E segment 18kb;

[0017] (2) The B and E segments of the genome were directly cloned into the p15A-amp vector to obtain recombinant plasmids p15A-amp-PRV-B and p15A-amp-PRV-E;

[0018] (3) Using the Bartha K61 genome as a template, small fragments with a length of less than or equal to 3kb were amplified by PCR. Then, the obtained small fragments were assembled with the p15A-amp vector using ExoCET multi-fragment assembly to obtain recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-C, p15A-amp-PRV-F and p15A-amp-PRV-G, which contain the A, C, F and G segments of the genome, respectively.

[0019] (4) Recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E were digested with enzymes to obtain PRV-A, PRV-B, PRV-C, PRV-F, PRV-G, and PRV-E. Each of the PRV-A fragment and the PRV-E fragment, the PRV-B fragment and the PRV-C fragment, and the PRV-F fragment and the PRV-G fragment all contain a 50bp homologous arm. Then, in vitro recombination was performed by Gibson with the BAC-cm vector containing the corresponding 50bp homologous arms according to the combination of EA, BC, and FG to obtain recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG.

[0020] According to a preferred embodiment of the present invention, in step (1), the cutting is specifically performed as follows: first, the Bartha K61 genome is cut into segments A (26kb), B (12kb), C (30kb), D (51kb), and E (18kb) in the order of 5'→3' using BglⅡ enzyme, and then segment D is cut into segments F (32kb) and G (19kb) using SwaI enzyme.

[0021] According to a preferred embodiment of the present invention, in step (3), adjacent small fragments are assembled using ExoCET multi-fragment assembly, wherein each adjacent small fragment contains a 50bp homologous arm, and assembly is performed through the homologous arm; in the PCR amplification, segment A of the genome contains 8 3kb small fragments and 1 2kb small fragment, segment C contains 10 3kb small fragments, segment F contains 10 3kb small fragments and 1 2kb small fragment, and segment G contains 6 3kb small fragments and 1 1kb small fragment.

[0022] According to a preferred embodiment of the present invention, in step (4), PacI and PmeI enzymes are used to digest the recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E.

[0023] According to a preferred embodiment of the present invention, the host cell is African green monkey kidney cell (Vero cell).

[0024] The application of the reverse genetics operating system for cloning and rescuing the genome of the pseudorabies virus Bartha K61 vaccine strain in the rescue of pseudorabies virus Bartha K61.

[0025] According to a preferred embodiment of the present invention, the specific method of the application is as follows:

[0026] 1) By using Redαβ homologous recombination, 2kb homologous arms were introduced at both ends of the PRV gene fragments in the recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG, so that the three fragments EA, BC, and FG had 2kb of overlapping sequences at their ends, resulting in recombinants BAC-cm-PRV-EA-2kHA, BAC-cm-PRV-BC-2kHA, and BAC-cm-PRV-FG-2kHA;

[0027] 2) The recombinants BAC-cm-PRV-EA-2kHA, BAC-cm-PRV-BC-2kHA and BAC-cm-PRV-FG-2kHA were digested with PacI enzyme to obtain the digested products PRV-EA-2kHA, PRV-BC-2kHA and PRV-FG-2kHA fragments. The digested products were then transfected into Vero cells to rescue the pseudorabies virus Bartha K61.

[0028] The application of the reverse genetics operating system for segmented cloning and rescue of the pseudorabies virus Bartha K61 vaccine strain in the development of genetically engineered vaccines.

[0029] All steps not described in detail in this invention are performed in accordance with existing technology.

[0030] Technical features and beneficial effects of the present invention:

[0031] 1. This invention relates to a reverse genetics operating system for the Bartha K61 pseudorabies virus vaccine strain. Using ExoCET cloning and assembly technology, the Bartha K61 genome was successfully divided into six segments (A segment 26kb, B segment 12kb, C segment 30kb, F segment 32kb, G segment 19kb, and E segment 18kb), which were then assembled into three recombinant plasmids: BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG. These three recombinant plasmids, after PacI linearization, can be co-transfected into African green monkey kidney cells to rescue the Bartha K61 virus. Furthermore, the infectivity of the rescued Bartha K61 virus is comparable to that of the vaccine strain. Therefore, this reverse genetics operating system can provide an excellent vector for creating live vector vaccines related to swine disease prevention and control, and can be used to develop Bartha K61 recombinant genetic engineering vector vaccines expressing relevant swine disease antigen genes.

[0032] 2. This invention is the first to successfully clone and rescue a large pseudorabies virus, Bartha K61, with a genome length of 137kb, providing ideas and methods for cloning and rescuing large viral genomes. Attached Figure Description

[0033] Figure 1 The segmentation scheme and experimental procedure for the first segmented cloning;

[0034] In the diagram, a represents the segmentation scheme, and b represents the experimental procedure.

[0035] Figure 2 Electrophoresis results for recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-D, and p15A-amp-PRV-E;

[0036] In the figure, lane 1 represents the DNA molecular weight standard, and the correct clones are marked in bold italics.

[0037] Figure 3 The segmentation scheme and experimental procedure for the second segmented cloning;

[0038] In the diagram, a represents the segmentation scheme, and b represents the experimental procedure.

[0039] Figure 4 Electrophoresis results for recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-C, p15A-amp-PRV-F, and p15A-amp-PRV-G;

[0040] In the figures, in a) lane 1 represents the DNA molecular weight standard, and lanes 2-10 represent the electrophoresis results of p15A-amp-PRV-A, with the correct clones marked in bold italics; in b) lane 1 represents the DNA molecular weight standard, and lanes 2-10 represent the electrophoresis results of p15A-amp-PRV-C, with the correct clones marked in bold italics; in c) lane 1 represents the DNA molecular weight standard, and lanes 2-10 represent the electrophoresis results of p15A-amp-PRV-F, with the correct clones marked in bold italics; and in d) lane 1 represents the DNA molecular weight standard, and lanes 2-10 represent the electrophoresis results of p15A-amp-PRV-G, with the correct clones marked in bold italics.

[0041] Figure 5 A flowchart illustrating the strategy for assembling Bartha K61 genome fragments;

[0042] In the figure, Figure a shows the recombinant plasmid BAC-cm-PRV; Figure b shows the recombinant plasmids BAC-cm-PRV-ABC and BAC-cm-PRV-FGE; Figure c shows the recombinant plasmids BAC-cm-PRV-AB, BAC-cm-PRV-CF, BAC-cm-PRV-GE, or BAC-cm-PRV-EA, BAC-cm-PRV-BC and BAC-cm-PRV-FG.

[0043] Figure 6 Electrophoresis results for recombinant plasmids BAC-cm-PRV, BAC-cm-PRV-ABC, BAC-cm-PRV-FGE, BAC-cm-PRV-AB, BAC-cm-PRV-CF, BAC-cm-PRV-GE, BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG;

[0044] In the figures, in a) lane 1 represents the DNA molecular weight standard, and lanes 2-12 represent the BAC-cm-PRV digestion results (no correct clones found); in b) lane 1 represents the DNA molecular weight standard, and lanes 2-12 represent the BAC-cm-PRV-ABC digestion results (no correct clones found); in c) lane 1 represents the DNA molecular weight standard, and lanes 2-12 represent the BAC-cm-PRV-FGE digestion results (no correct clones found); in d) lane 1 represents the DNA molecular weight standard, and lanes 2-11 represent the BAC-cm-PRV-AB digestion results (the clones marked in bold italics are correct clones); lanes 12-23 represent the BAC-cm-PRV-FGE digestion results. In Figure e, lane 1 represents the DNA molecular weight standard, and lanes 2-8 represent the BAC-cm-PRV-GE digestion results, with no correct clones. Lanes 2-36 represent the BAC-cm-PRV-EA digestion results, with the correct clones marked in bold italics. In Figure f, lane 1 represents the DNA molecular weight standard, and lanes 2-8 represent the BAC-cm-PRV-BC digestion results, with the correct clones marked in bold italics. In Figure g, lane 1 represents the DNA molecular weight standard, and lanes 2-8 represent the BAC-cmp-PRV-FG digestion results, with the correct clones marked in bold italics.

[0045] Figure 7 Flowcharts illustrating two strategies for rescuing Bartha K61 vaccine strains of pseudorabies virus;

[0046] In the diagram, diagram a represents strategy one, and diagram b represents strategy two.

[0047] Figure 8 Electrophoresis results for recombinants BAC-cm-PRV-EA-2kHA, BAC-cm-PRV-BC-2kHA, and BAC-cm-PRV-FG-2kHA;

[0048] In Figure a, lane 1 represents the DNA molecular weight standard, lanes 2-8 represent the enzyme digestion results of BAC-cm-PRV-EA-2kHA, lanes 9-15 represent the enzyme digestion results of BAC-cm-PRV-BC-2kHA, and lanes 16-22 represent the enzyme digestion results of BAC-cm-PRV-FG-2kHA. The clones marked in bold italics are the correct clones. In Figure b, lane 1 represents the DNA molecular weight standard, and lanes 2-10 represent the enzyme digestion results of p15A-cm-PRV-E-2kHA. The clones marked in bold italics are the correct clones.

[0049] Figure 9 Schematic diagram and results of the Bartha K61 vaccine strain for saving pseudorabies virus;

[0050] In the figure, a) shows the connection method of combination one; b) shows the connection method of combination two; c) shows the Vero cell control; d) shows the lesion results of co-transfecting Vero cells with combination one; and e) shows the lesion results of co-transfecting Vero cells with combination two.

[0051] Figure 10 This invention provides a growth curve verification for the pseudorabies virus Bartha K61 obtained through the rescue method of this invention. Detailed Implementation

[0052] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0053] Example 1: Cloning of the Bartha K61 genome

[0054] 1. Direct cloning

[0055] The Bartha K61 genome (Genbank ID JF797217.1) was analyzed using the software SnapGene. Its genome length is approximately 137kb, with a GC content of 74%. It contains repetitive sequences at both ends. One end of the UL region contains an 80bp repetitive sequence with a 361bp gap between the terminal and internal repetitive sequences. The other end of the US region contains approximately 16kb of repetitive sequences with a 5kb gap between the terminal and internal repetitive sequences. It cannot be directly cloned.

[0056] 2. First segmented cloning

[0057] The p15A-amp vector used for direct cloning of ExoCET was obtained by PCR amplification using the primers in Table 1. For example... Figure 1As shown in (a), the Bartha K61 genome was cut into five segments in a 5'→3' sequence using the BglII enzyme, named segments A, B, C, D, and E, with lengths of 26kb, 12kb, 30kb, 51kb, and 18kb, respectively. Using ExoCET cloning technology, each segment was ligated into the p15A-amp vector to construct recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E containing the complete genome of the pseudorabies virus Bartha K61 vaccine strain. The procedure is as follows: Figure 1 As shown in (b).

[0058] Recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, and p15A-amp-PRV-D were identified by restriction endonuclease NcoⅠ, and p15A-amp-PRV-E was identified by restriction endonuclease BamHI. The results were analyzed by agarose gel electrophoresis. Figure 2 As shown.

[0059] Depend on Figure 2 It was found that only segments B and E of the Bartha K61 pseudorabies virus vaccine strain were successfully cloned, while segments A, C, and D were longer. The concentration of the Bartha K61 genome fragments after enzyme digestion was measured and found to be low, which may have led to the failure of direct cloning.

[0060] Table 1. Primers used in the embodiments of this invention

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] 3. Second segmented cloning

[0068] Following the method in step 2, the Bartha K61 genome was cut into five segments. Then, the SwaI enzyme was used to cut segment D into segments F and G, resulting in the Bartha K61 genome being cut into six segments: segment A (26kb), segment B (12kb), segment C (30kb), segment F (32kb), segment G (19kb), and segment E (18kb).

[0069] The recombinant plasmids p15A-amp-PRV-B and p15A-amp-PRV-E can be obtained according to the method in step 2.

[0070] Using the PRV genome as a template, and employing the primers listed in Table 1, high-fidelity DNA polymerase was used to amplify small fragments of 3 kb or less in length, with each adjacent fragment containing a 50 bp homologous arm. The PCR-amplified fragments were then assembled into the p15A-amp vector using ExoCET multi-fragment assembly to obtain recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-C, p15A-amp-PRV-F, and p15A-amp-PRV-G, each containing fragments A, C, F, and G, respectively. The procedure is as follows: Figure 3 As shown in (b). In the PCR amplification, segment A of the genome contains 8 3kb fragments and 1 2kb fragment, segment C contains 10 3kb fragments, segment F contains 10 3kb fragments and 1 2kb fragment, and segment G contains 6 3kb fragments and 1 1kb fragment.

[0071] Recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-C, and p15A-amp-PRV-F were identified by restriction endonuclease NcoI, and p15A-amp-PRV-G was identified by restriction endonuclease AfeI. The results were analyzed by agarose gel electrophoresis. Figure 4 As shown.

[0072] Depend on Figure 4 It can be seen that the plasmid digestion results of segments A, C, F and G of the Bartha K61 genome were all correctly cloned, that is, the full-length Bartha K61 genome of 137kb can be cloned in the following segmentation method: segment A 26kb, segment B 12kb, segment C 30kb, segment F 32kb, segment G 19kb and segment E 18kb.

[0073] Example 2: Construction of recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG

[0074] 1. Recombinant p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E were digested with PacI and PmeI to obtain PRV-A, PRV-B, PRV-C, PRV-F, PRV-G, and PRV-E fragments. Among these, there are 50 bp homologous arms between PRV-A and PRV-B, between PRV-B and PRV-C, between PRV-C and PRV-F, between PRV-F and PRV-G, between PRV-G and PRV-E, and between PRV-A and PRV-E.

[0075] The full-length Bartha K61 genome was assembled using three strategies:

[0076] Strategy 1: PRV-A, PRV-B, PRV-C, PRV-F, PRV-G, and PRV-E fragments are directly recombined to form a single plasmid;

[0077] Strategy 2: Combine plasmids in the order of A+B+C and F+G+E to form two recombinant plasmids;

[0078] Strategy 3: Combine plasmids in the order of A+B, C+F, and G+E to form three recombinant plasmids, or combine them in the order of E+A, B+C, and F+G to form three recombinant plasmids. Specific strategies and procedures are as follows: Figure 5 As shown.

[0079] 2. First assembly of Bartha K61 genome fragments

[0080] p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E were digested with PacI and PmeI enzymes. The resulting digested fragments were then integrated into the BAC-cm vector containing the corresponding homologous arms at the ends using Gibson in vitro recombination technology according to strategy one, to obtain the recombinant plasmid BAC-cm-PRV.

[0081] 3. Second assembly of Bartha K61 genome fragments

[0082] p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E were digested with PacI and PmeI enzymes. The resulting digested fragments were then integrated into the BAC-cm vector containing the corresponding homologous arms at the ends using Gibson in vitro recombination technology according to strategy two, resulting in recombinant plasmids BAC-cm-PRV-ABC and BAC-cm-PRV-FGE.

[0083] 3. Third assembly of Bartha K61 genome fragments

[0084] p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E were digested with PacI and PmeI enzymes. The resulting digested fragments were then integrated into the BAC-cm vector containing the corresponding homologous arms at the ends using Gibson in vitro recombination technology according to strategy three, to obtain recombinant plasmids BAC-cm-PRV-AB, BAC-cm-PRV-CF, and BAC-cm-PRV-GE, or recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG.

[0085] 5. Recombinant plasmids BAC-cm-PRV, BAC-cm-PRV-ABC, BAC-cm-PRV-FGE, BAC-cm-PRV-AB, BAC-cm-PRV-CF, and BAC-cm-PRV-GE were identified by digestion with restriction endonuI; BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG were identified by digestion with restriction endonuI. The results were analyzed by agarose gel electrophoresis. Figure 6 As shown.

[0086] The electrophoresis results of the recombinant plasmid BAC-cm-PRV formed by combining strategies A+B+C+F+G+E showed that all of them were self-circularized within the BAC-cm vector, thus assembly failed. Figure 6 a).

[0087] The electrophoresis results of the two recombinant plasmids BAC-cm-PRV-ABC and BAC-cm-PRV-FGE formed by combining A+B+C and F+G+E in strategy two still show some loss of restriction enzyme bands and positional differences compared with the theoretical electrophoresis diagram. Figure 6 (b and c).

[0088] Strategy 3, using combinations of A+B, C+F, and G+E to form three recombinant plasmids: BAC-cm-PRV-AB, BAC-cm-PRV-CF, and BAC-cm-PRV-GE, yielded the correct clone in electrophoresis. However, the AB combination failed to find the correct clone, with varying degrees of restriction band loss and positional differences observed. Figure 6 d).

[0089] Strategy 3, using the combinations E+A, B+C, and F+G in that order to form three recombinant plasmids: BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG, showed that the correct clones were found for all three combinations: EA, BC, and FG. Figure 6 e.g.

[0090] 6. In this embodiment, recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG containing the complete genome of the pseudorabies virus Bartha K61 vaccine strain were successfully obtained. These three plasmids constitute the reverse genetic operating system of the pseudorabies virus Bartha K61 vaccine strain. After transfection into host cells, they can save the pseudorabies virus Bartha K61.

[0091] Example 3: Strategies for rescuing the Bartha K61 vaccine strain of pseudorabies virus

[0092] 1. Two strategies were adopted for the rescue of the Bartha K61 vaccine strain of pseudorabies virus:

[0093] Strategy 1: The recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG were digested with PacI to obtain PRV-EA, PRV-BC, and PRV-FG fragments. The digestion products were transfected into host cells, and the fragments were ligated to form the complete Bartha K61 genome under the mediation of the cell's own homologous recombination system.

[0094] Strategy 2: The recombinant plasmids BAC-cm-PRV-A, BAC-cm-PRV-E, BAC-cm-PRV-BC, and BAC-cm-PRV-FG were digested with PacI to obtain PRV-A, PRV-E, PRV-BC, and PRV-FG fragments. The digestion products were transfected into host cells, and the fragments were ligated to form the complete Bartha K61 genome under the mediation of the cell's own homologous recombination system.

[0095] 2. For example Figure 7As shown, following strategy one, using pR6K-oriT-phiC31-apra (Wang H,Li Z,Jia R,HouY,Yin J,Bian X,et al.RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression.Nature Protocols.2016;11(7):1175-90.) as a template, the apra resistance fragment was amplified by PCR using the primers in Table 1; then, using the BarthaK61 genome as a template, a 2kb homologous arm fragment was obtained by PCR using the primers in Table 1, and then the resistance fragment was ligated to the homologous arm fragment by tandem PCR to obtain the apra-2kHA fragment.

[0096] BAC-cm-PRV-EA / BC / FG was electroporated into E. coli GB08-red. Redαβ homologous recombination was then performed to introduce 2kb homologous arms at both ends of the PRV gene fragments in the recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG obtained in Example 2, so that the three fragments EA, BC, and FG had 2kb of overlapping sequences at their ends, resulting in recombinants BAC-cm-PRV-EA-2kHA, BAC-cm-PRV-BC-2kHA, and BAC-cm-PRV-FG-2kHA.

[0097] 3. For example Figure 7As shown, following strategy two, p15A-cm-tetO-tetR-hyg-ccdB (Wang H, Li Z, Jia R, Hou Y, Yin J, Bian X, et al. RecET direct cloning and Redab recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nature) Using Protocols.2016;11(7):1175-90. as a template, the vector fragment p15A-cm was amplified by PCR using the primers in Table 1; then, using the BarthaK61 genome as a template, a 2kb homologous arm fragment was obtained by PCR, and then the vector fragment and the homologous arm fragment were ligated together by tandem PCR to obtain the fragment p15A-cm-2kHA. The plasmid p15A-amp-PRV-E was digested with enzymes, and the digested product and the fragment p15A-cm-2kHA were co-transformed into E. coli GB05-dir. p15A-cm-PRV-E-2kHA was obtained by RecET homologous recombination technology.

[0098] 4. The recombinants BAC-cm-PRV-E-2kHA, BAC-cm-PRV-EA-2kHA, BAC-cm-PRV-BC-2kHA, and BAC-cm-PRV-FG-2kHA were identified by digestion with the restriction endonuclease Nco I, and p15A-cm-PRV-E-2kHA was identified by digestion with the restriction endonuclease Not I. Agarose gel electrophoresis analysis was then performed, and the electrophoresis results are shown below. Figure 8 .

[0099] Depend on Figure 8 It can be seen that the recombinant plasmids of both strategies were successfully constructed.

[0100] Example 4: Rescue of the Bartha K61 vaccine strain for pseudorabies virus

[0101] 1. The recombinant plasmids BAC-cm-PRV-EA-2kHA, BAC-cm-PRV-BC-2kHA, and BAC-cm-PRV-FG-2kHA from combination one were digested with PacI to obtain PRV-EA-2kHA, PRV-BC-2kHA, and PRV-FG-2kHA fragments. The recombinant plasmids p15A-cm-PRV-E-2kHA, BAC-cm-PRV-A, BAC-cm-PRV-BC-2kHA, and BAC-cm-PRV-FG-2kHA from combination two were digested with PacI to obtain PRV-E, PRV-A, PRV-BC-2kHA, and PRV-FG-2kHA fragments. The enzyme digestion products were transfected into Vero cells at a density of 90%, and incubated for 6 hours. Then, DMEM medium containing 2% fetal bovine serum was added. 48 hours after transfection, both products underwent three freeze-thaw cycles and were re-seeded into well-growing Vero cells up to the F3 generation. Normal Vero cells were used as a control. The results were observed as follows: Figure 9 As shown.

[0102] Depend on Figure 9 It can be seen that over 90% of the cells in combination one showed cytopathic effects, while combination two and the control group did not show any cytopathic effects. This indicates that combination one successfully rescued Bartha K61 pseudorabies virus, while combination two failed. Recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG, containing only the complete genome of the Bartha K61 pseudorabies virus vaccine strain, along with host cells, can constitute a reverse genetic operating system for the Bartha K61 pseudorabies virus vaccine strain, achieving the goal of rescuing Bartha K61 pseudorabies virus.

[0103] 2. Growth kinetic curves were determined from seven different pseudorabies viruses Bartha K61 (B157, B158, B167, B168, B257, B258 and B267) obtained by the rescue method of this invention.

[0104] The specific method is as follows: Seven different pseudorabies viruses, Bartha K61, obtained through the rescue method of this invention, were inoculated into Vero cells. Samples were then taken periodically and quantitatively, and the TCID50 of the obtained virus samples was determined and compared with that of the parent wild-type Bartha K61 virus. The results are as follows: Figure 10 As shown.

[0105] Depend on Figure 10It can be seen that the pseudorabies virus Bartha K61 obtained by the rescue method of the present invention exhibits the same growth kinetic level as the wild pseudorabies virus Bartha K61 (Bartha), indicating that the cloning and rescue method of the present invention does not have a significant impact on the replication of the recombinant virus.

Claims

1. A reverse genetics operating system for segmented cloning and rescue of the pseudorabies virus Bartha K61 vaccine strain genome, characterized in that, The system includes: recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG containing the complete genome of the pseudorabies virus Bartha K61 vaccine strain, and host cells expressing the complete genome of the pseudorabies virus Bartha K61 vaccine strain. The construction methods of the recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG are as follows: (1) The Bartha K61 genome was cut into six segments: segment A (26kb), segment B (12kb), segment C (30kb), segment F (32kb), segment G (19kb), and segment E (18kb); Specifically, the cutting process involves: first using... Bgl Enzyme II cut the Bartha K61 genome into segments A (26kb), B (12kb), C (30kb), D (51kb), and E (18kb), and then SwaI enzyme cut segment D into segments F (32kb) and G (19kb). (2) The B and E segments of the genome were directly cloned into the p15A-amp vector to obtain recombinant plasmids p15A-amp-PRV-B and p15A-amp-PRV-E; (3) Using the Bartha K61 genome as a template, small fragments with a length of less than or equal to 3kb were amplified by PCR. Then, the obtained small fragments were assembled with the p15A-amp vector using ExoCET multi-fragment assembly to obtain recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-C, p15A-amp-PRV-F and p15A-amp-PRV-G, which contain the A, C, F and G segments of the genome, respectively. (4) After digesting the recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E, PRV-A, PRV-B, PRV-C, PRV-F, PRV-G, and PRV-E fragments were obtained. Each of the PRV-A and PRV-E fragments, the PRV-B and PRV-C fragments, and the PRV-F and PRV-G fragments contained a 50bp homologous arm. Then, the recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG were obtained by in vitro recombination with the BAC-cm vector containing the corresponding 50bp homologous arms according to the combination of EA, BC, and FG.

2. The reverse genetics operating system as described in claim 1, characterized in that, In step (3), adjacent small segments are assembled using ExoCET multi-segment assembly. Each of the adjacent small segments contains a 50 bp homologous arm, which is used for assembly.

3. The reverse genetics operating system as described in claim 1, characterized in that, In step (4), PacI and PmeI enzymes were used to digest the recombinant plasmids p15A-amp-PRV-A, p15A-amp-PRV-B, p15A-amp-PRV-C, p15A-amp-PRV-F, p15A-amp-PRV-G, and p15A-amp-PRV-E.

4. The reverse genetics operating system as described in claim 1, characterized in that, The host cells were African green monkey kidney cells.

5. The application of the reverse genetics operating system for genome segmentation cloning and rescue of the pseudorabies virus Bartha K61 vaccine strain as described in claim 1 in the rescue of pseudorabies virus Bartha K61.

6. The application as described in claim 5, characterized in that, The specific method of the application is as follows: 1) By using Redαβ homologous recombination, 2 kb homologous arms were introduced at both ends of the PRV gene fragments in the recombinant plasmids BAC-cm-PRV-EA, BAC-cm-PRV-BC, and BAC-cm-PRV-FG, so that the three fragments EA, BC, and FG had 2 kb of overlapping sequences at their ends, resulting in recombinants BAC-cm-PRV-EA-2kHA, BAC-cm-PRV-BC-2kHA, and BAC-cm-PRV-FG-2kHA; 2) The recombinants BAC-cm-PRV-EA-2kHA, BAC-cm-PRV-BC-2kHA and BAC-cm-PRV-FG-2kHA were digested with PacI enzyme to obtain the digested products PRV-EA-2kHA, PRV-BC-2kHA and PRV-FG-2kHA fragments. The digested products were then transfected into Vero cells to rescue the pseudorabies virus Bartha K61.

7. The application of the reverse genetics operating system for genome segmentation cloning and rescue of the pseudorabies virus Bartha K61 vaccine strain as described in claim 1 in the development of genetically engineered vaccines.

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