Construction and application of recombinant pseudorabies virus strain capable of stably and efficiently expressing green fluorescent protein

Abstract

The invention provides construction and application of a recombinant pseudorabies virus strain capable of stably and efficiently expressing green fluorescent protein, which is characterized in that green fluorescent protein (EGFP) genes are respectively inserted into different positions of a pseudorabies virus variant double-gene deletion vaccine strain (JS-2012-gE / gI) by using a homologous recombination method. Finally, the recombinant virus JS-2012-gE / gI-VP1 / 2-EGFP has good genetic stability, the EGFP gene can be stably and efficiently expressed, and the proliferation rate of the EGFP gene on a cell is equivalent to that of a parent virus, so that the VP1 / 2 gene is an optimal exogenous gene insertion site. The classical swine fever virus E2 gene, the parvovirus VP2 gene, the cap gene of the circovirus type 2 and the VP1 gene of the foot-and-mouth disease virus can be subsequently replaced or inserted into an EGFP expression cassette in an independent or combined manner to construct different types of multi-combined vaccines, so that the multi-combined vaccine has extremely high research value and platform significance.

Description

Technical Field

[0001] The present invention belongs to the field of animal medicine, and specifically relates to a platform-based research and development of a porcine pseudorabies virus gene-deleted vaccine strain, and especially to the construction and application of a recombinant pseudorabies virus strain that stably and efficiently expresses green fluorescent protein. Background Art

[0002] Pseudorabies virus (PRV), a double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily of the Herpesviridae family, infects pigs and causes pseudorabies (Aujeszky's disease), causing significant economic losses to the global swine industry. Due to its genome's ability to accommodate a large number of exogenous genes and its wide host range, PRV has become an important tool for live viral vector vaccines and neural circuit tracing research with advances in genetic engineering. The selection of insertion sites is a key factor in the success of live viral vector vaccine development. Previous studies have shown that replication-non-essential regions of the PRV genome (such as the TK gene and gE / gI gene regions) are often selected as insertion sites for exogenous genes. However, recombinant viruses with different insertion sites exhibit varying replication efficiency, genetic stability, and exogenous gene expression in cells. Furthermore, the selection of insertion sites must balance the regulation of exogenous gene expression with the synergy between the viral genes themselves. However, systematic studies of different insertion sites in the PRV genome remain limited.

[0003] From a scientific perspective, the PRV genome is highly complex and functionally redundant, with distinct gene regions playing significantly different roles in the viral life cycle. The selection of insertion sites for exogenous genes must not only consider viral replication and immunogenicity, but also the impact of the insertion site on the overall stability of the viral genome. Existing experimental data also confirm that although commonly used insertion sites in the PRV genome (such as the gE, gI, and TK gene regions) have been widely used in the development of gene-deleted vaccines or recombinant viral vectors, the effects of different sites on viral biological properties (such as replication efficiency, virulence, and immunogenicity) vary greatly. Finding or establishing a stable insertion site research platform remains an uncertain research area. Summary of the Invention

[0004] In order to solve the above problems, the present application first provides a group of recombinant viruses with different exogenous genes based on the parental virus JS-2012-ΔgE / gI. The specific insertion sites of the recombinant viruses are shown in Table 1:

[0005] Table 1 Specific insertion sites on each gene

[0006]

[0007]

[0008] Furthermore, the recombinant virus provided by the present application specifically inserts the green fluorescent protein gene (EGFP) into different positions of the pseudorabies virus variant double gene deletion vaccine strain (JS-2012-△gE / gI), and the insertion sites are located after the CDS regions of 9 genes: gE, VP1 / 2, gB, gC, UL26, gD, gG, TK and gM;

[0009] The 9 recombinant viruses obtained were further named JS-2012-△gE / gI-gE-EGFP, JS-2012-△gE / gI-VP1 / 2-EGFP, JS-2012-△gE / gI-gB-EGFP, JS-2012-△gE / gI-gC-EGFP, JS-2012-△gE / gI-UL26-EGFP, JS-2012-△gE / gI-gD-EGFP, JS-2012-△gE / gI-gG-EGFP, JS-2012-△gE / gI-TK-EGFP and JS-2012-△gE / gI-gM-EGFP.

[0010] More preferably, the present application provides a recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP, which shows good genetic stability during continuous passage, and the EGFP gene can also be stably and efficiently expressed, and the proliferation rate in cells is also comparable to that of the parent virus. Therefore, the VP1 / 2 gene is the optimal site for exogenous gene insertion.

[0011] Furthermore, the present invention provides a monoclonal antibody that can specifically recognize the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP, wherein the light chain variable region amino acid sequence of the monoclonal antibody is shown in SEQ ID NO: 2, the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 3, the light chain CDR1 amino acid sequence is shown in SEQ ID NO: 4, the light chain CDR2 amino acid sequence is shown in SEQ ID NO: 5, the light chain CDR3 amino acid sequence is shown in SEQ ID NO: 6, the heavy chain CDR1 amino acid sequence is shown in SEQ ID NO: 7, the heavy chain CDR2 amino acid sequence is shown in SEQ ID NO: 8, and the heavy chain CDR3 amino acid sequence is shown in SEQ ID NO: 9.

[0012] Furthermore, the present invention provides a platform for constructing a recombinant pseudorabies virus vaccine strain, wherein the platform is the recombinant virus JS-2012-ΔgE / gI-VP1 / 2-EGFP.

[0013] Furthermore, using the recombinant pseudorabies virus strain vaccine strain construction platform, the E2 gene of classical swine fever virus, the VP2 gene of parvovirus, the cap gene of circovirus type 2, and the VP1 gene of foot-and-mouth disease virus are replaced or inserted into the EGFP expression frame individually or in combination to construct different types of multi-component vaccines.

[0014] Furthermore, the present invention provides a use of a recombinant virus JS-2012-ΔgE / gI-VP1 / 2-EGFP, a monoclonal antibody, and a vaccine construction platform in the preparation of a drug for treating or preventing diseases caused by pseudorabies virus.

[0015] Beneficial effects

[0016] In this study, the green fluorescent protein gene (EGFP) was inserted into different positions of the pseudorabies virus variant double gene deletion vaccine strain (JS-2012-△gE / gI) using the homologous recombination method. The insertion sites were located after the CDS regions of nine genes, including gE, VP1 / 2, gB, gC, UL26, gD, gG, TK and gM. Ultimately, nine recombinant viruses were successfully constructed and named JS-2012-△gE / gI-gE-EGFP, JS-2012-△gE / gI-VP1 / 2-EGFP, JS-2012-△gE / gI-gB-EGFP, JS-2012-△gE / gI-gC-EGFP, JS-2012-△gE / gI-UL26-EGFP, JS-2012-△gE / gI-gD-EGFP, JS-2012-△gE / gI-gG-EGFP, JS-2012-△gE / gI-TK-EGFP, and JS-2012-△gE / gI-gM-EGFP. Each of the nine recombinant viruses was blind-passaged on Vero cells for 20 passages to verify their genetic stability. The replication ability, EGFP protein expression, and other biological properties of the fifth-passage recombinant viruses were then compared and analyzed. The results showed that the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP lost fluorescence at the beginning of continuous passaging, and JS-2012-△gE / gI-gD-EGFP lost fluorescence after continuous passaging to the 12th generation. PCR and sequencing verification showed that the target genes were indeed lost to varying degrees and had poor genetic stability. This shows that gC, UL26, and gD genes are not suitable as sites for exogenous gene insertion. The recombinant viruses JS-2012-△gE / gI-gE-EGFP, JS-2012-△gE / gI-gB-EGFP, JS-2012-△gE / gI-gG-EGFP, JS-2012-△gE / gI-TK-EGFP, and JS-2012-△gE / gI-gM-EGFP all exhibited good genetic stability during serial passage, and the EGFP gene was stably and efficiently expressed. However, since their cell proliferation rates were significantly lower than those of the parental virus, the gE, gB, gG, TK, and gM genes were not optimal sites for exogenous gene insertion. The recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP exhibited good genetic stability during serial passage, and the EGFP gene was stably and efficiently expressed. Its cell proliferation rate was comparable to that of the parental virus, indicating that the VP1 / 2 genes were optimal sites for exogenous gene insertion.

[0017] This study, by constructing multiple recombinant viruses with different insertion sites and combining them with in vitro cell experiments, systematically revealed the mechanisms by which different insertion sites influence viral phenotypes. This study is expected to fill a research gap in this field and deepen our understanding of the functional modularity of the PRV genome. From a practical perspective, identifying efficient and safe insertion sites has a direct impact on PRV vaccine development. First, optimizing the insertion site can improve the safety of gene-deletion vaccines. Second, the choice of insertion site may influence the expression level of exogenous antigens and the strength of the immune response. For example, certain gene regions (such as those downstream of the promoter of early-expressing genes) may be more conducive to efficient transcription of exogenous genes, thereby enhancing the multivalent immune response of recombinant vaccines. Furthermore, in viral vector development, the stability of the insertion site directly determines the long-term expression capacity of exogenous genes. By selecting sites with high genetic stability, the risk of gene rearrangement or loss during recombinant virus passage can be reduced, providing technical support for the use of PRV as a vaccine vector (e.g., for delivering African swine fever virus antigens). Furthermore, this study has important implications for the sustainable development of animal husbandry. Pseudorabies prevention and control is a core component of swine health management, and the optimization of genetically engineered vaccines can reduce the potential for latent infection or reversion to virulence caused by traditional vaccines (such as attenuated vaccines). By screening for optimal insertion sites, the development of a new generation of marker vaccines (DIVA vaccines) can be promoted, effectively combining disease elimination and monitoring. Furthermore, as a model pathogen for neurotropic viruses, the optimization of PRV's vector system can provide cross-species references for gene therapy research with human herpes viruses (such as HSV-1), with potential translational medical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Activity verification of each transfer vector;

[0019] Figure 2 Identification results of each co-transfected virus after purification;

[0020] Figure 3 One-step growth curves of each recombinant virus and parental virus;

[0021] Figure 4 Plaque morphology of each recombinant virus and parental virus;

[0022] Figure 5 Statistics of plaque diameters of each recombinant virus and parental virus;

[0023] Figure 6 Fluorescence images of JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP during passage Figure 7 PCR identification of JS-2012-△gE / gI-gC-EGFP co-transfected virus and 4th generation virus;

[0024] Figure 8 PCR identification of JS-2012-△gE / gI-UL26-EGFP co-transfected virus and 4th generation virus;

[0025] Figure 9 Sequencing analysis of JS-2012-△gE / gI-gC-EGFP PCR miscellaneous bands;

[0026] Figure 10 Sequencing analysis of JS-2012-△gE / gI-UL26-EGFP PCR miscellaneous bands;

[0027] Figure 11 Sequencing analysis of JS-2012-△gE / gI-UL26-EGFP PCR bands, where M2: Trans 2K DNA Marker; 1-20: 1-20th generation recombinant virus samples; 21: positive control; 22: negative control;

[0028] Figure 12 Sequencing analysis of JS-2012-△gE / gI-gD-EGFP PCR miscellaneous bands;

[0029] Figure 13 3-11 PCR identification of 20 generations of each recombinant virus, M1: Trans 5K DNA Marker; M2: Trans 2K DNA Marker; 1-20: 1-20 generations of recombinant virus samples; 21: positive control; 22: negative control;

[0030] Figure 14 The results of viral gene sequencing and target gene comparison of each recombinant virus generation FI, F10, and F20;

[0031] Figure 15Western blot results of EGFP protein expression of the 20th generation of each recombinant virus, M: protein molecular weight standard; 1: Western blot results of EGFP protein expression of JS-2012-ΔgE / gI-VP1 / 2-EGFP; 2: Western blot results of EGFP protein expression of JS-2012-ΔgE / gI-gE-EGFP; 3: Western blot results of EGFP protein expression of JS-2012-ΔgE / gI-gD-EGFP; 4: Western blot results of EGFP protein expression of JS-2012-ΔgE / gI-gG-EGFP; 5: Western blot results of EGFP protein expression of JS-2012-ΔgE / gI-gB-EGFP; 6: Western blot results of EGFP protein expression of JS-2012-ΔgE / gI-TK-EGFP blot results; 7: Western blot results of JS-2012-ΔgE / gI-gM-EGFP EGFP protein expression; 8: Positive control JS-2012-ΔgE / gI; 9: Negative control. DETAILED DESCRIPTION

[0032] The cell viruses used include Bhk cells, Vero cells, and the parental virus JS-2012-ΔgE / gI (Wu Tong, Guoxin Li, Chao Liang, Fei Liu, Qing Tian, Yanyun Cao, Lin Li, Xuchen Zheng, HaoZheng, Guangzhi Tong. A live, attenuated pseudorabies virus strain JS-2012 deleted for gE / gI protects against both classical and emerging strains. Antiviral Research 130 (2016) 110-117.), all of which are kept in this laboratory. Other reagents, instruments, and equipment are routine operations in this field and are selected according to specific circumstances. They are not the invention points of this application.

[0033] Example 1 Determination of the specific insertion site of the recombinant pseudorabies virus strain

[0034] Based on the applicant's previous research, it was finally determined that the insertion sites were selected on the gE gene, VP1 / 2 gene, gD gene, gG gene, gB gene, TK gene, gM gene, gC gene and UL26 gene of the parental virus JS-2012-ΔgE / gI. The specific insertion sites selected on the parental virus gene sequence are shown in Table 1:

[0035] Table 1 Specific insertion sites on each gene

[0036]

[0037] Based on the complete gene sequence of the PRV variant deletion strain JS-2012-ΔgE / gI, available in the laboratory, homology arms were designed upstream and downstream of each insertion site using Primer 5.0. The cloning vector pBluescript ll SK(+) was selected, and the target gene was a CMV+EGFP+SV40 sequence, both of which were maintained in the laboratory. The transfer vectors pBluescript ll SK(+)-gE-EGFP and pBluescript ll SK(+)-gG-EGFP, corresponding to the insertion sites on the gE and gG genes, were also maintained in the laboratory. The construction of transfer vectors corresponding to the insertion sites on the VP1 / 2 gene, gD gene, gB gene, TK gene, gM gene, gC gene and UL26 gene was handed over to Shanghai Saiheng Biotechnology Co., Ltd. and completed. They were respectively named pBluescript 11 SK(+)-VP1 / 2-EGFP, pBluescript 11 SK(+)-gD-EGFP, pBluescript 11 SK(+)-gB-EGFP, pBluescript 11 SK(+)-TK-EGFP, pBluescript 11 SK(+)-gM-EGFP, pBluescript 11 SK(+)-gC-EGFP and pBluescript 11 SK(+)-UL26-EGFP. The above vectors were all sequenced to verify their correctness. The nucleotide sequence of pBluescript 11 SK(+)-VP1 / 2-EGFP is shown in SEQ ID NO.1.

[0038] The specific left and right homology arm intervals on each transfer vector are shown in Table 2:

[0039] Table 2 Specific left and right homology arm intervals on each transfer vector

[0040]

[0041]

[0042] Sequencing verified that the transfer vector plasmids were successfully prepared. After expanded culture, the expression levels of each transfer vector were observed in BHK cells to verify the activity of each transfer vector. The transfection amount of each transfer vector plasmid was 2 μg. 125 μL of OPTI-MEM reagent and 5 μL of Lipo3000 reagent were mixed in an EP tube and transfected into BHK cells. The expression of the EGFP gene in each well was observed under a green fluorescence microscope to determine the activity of each transfection vector after transfection. The results are as follows Figure 1 As shown, it was confirmed that each transfer vector could be efficiently expressed in cells by constructing a specific eukaryotic expression vector and transforming it into cells.

[0043] To improve co-transfection efficiency, the transfer vectors were linearized. The pBluescript ll SK(+)-gE-EGFP and pBluescript ll SK(+)-gG-EGFP transfer vectors were linearized with XhoI restriction endonuclease, and the pBluescript ll SK(+)-VP1 / 2-EGFP, pBluescript ll SK(+)-gD-EGFP, pBluescript ll SK(+)-gB-EGFP, pBluescript ll SK(+)-TK-EGFP, pBluescript ll SK(+)-gM-EGFP, pBluescript ll SK(+)-gC-EGFP, and pBluescript ll SK(+)-UL26-EGFP transfer vectors were linearized with HindIII-HF restriction endonuclease. The JS-2012-ΔgE / gI genome was also extracted using the phenol-chloroform method.

[0044] 2 μg of linearized transfer vector and 2 μg of parental virus genome were co-transfected, and after collecting the co-transfected virus, the recombinant virus was purified using Vero cells. After each recombinant virus was completely purified, PCR identification of the recombinant virus was performed.

[0045] Based on the left and right homology arm sequences of each transfer vector, upstream and downstream primers were designed using Primer software. The upstream and downstream primers corresponding to the gE and gG insertion sites are kept in the laboratory. The specific primer names and sequences are shown in Table 3:

[0046] Table 3 Names and sequences of primers for identifying recombinant viruses

[0047]

[0048] The PCR reaction identification methods for each recombinant virus are divided into two types based on the identification results:

[0049] Recombinant viruses JS-2012-ΔgE / gI-gE-EGFP, JS-2012-ΔgE / gI-gB-EGFP, JS-2012-ΔgE / gI-gC-EGFP, JS-2012-ΔgE / gI-VP1 / 2-EGFP, and JS-2012-ΔgE / gI-UL26-EGFP were identified using LA Taq enzyme. The reaction system was 25 μL, and the amounts of each component are shown in Table 4:

[0050] Table 4 LA Taq enzyme identification system

[0051]

[0052] Recombinant viruses JS-2012-△gE / gI-gD-EGFP, JS-2012-△gE / gI-TK-EGFP, JS-2012-

[0053] ΔgE / gI-gG-EGFPH and JS-2012-ΔgE / gI-gM-EGFP were identified using 2×GC-rich Master Mix. The reaction system was 20 μL, and the amounts of each component were as shown in Table 5:

[0054] Table 5 2×GC-rich Master Mix identification system

[0055]

[0056] The PCR reaction conditions for identification of each recombinant virus were the same except for the annealing temperature and extension time, as shown in Table 6. The reaction conditions were as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, annealing for 30 s, extension at 72°C, and final extension at 72°C for 10 min, for a total of 35 reaction cycles.

[0057] Table 6 Annealing temperature and extension time of each recombinant virus PCR identification

[0058]

[0059]

[0060] The results are as follows Figure 2 As shown, all the identification bands were consistent with expectations, indicating that pure recombinant viruses were obtained.

[0061] Example 2 Analysis of biological characteristics of recombinant viruses

[0062] Analysis of virus growth characteristics

[0063] The fifth generation virus of each recombinant virus and the fifth generation virus of the parent virus were selected for comparison of toxicity titers. The specific operation is as follows:

[0064] (1) Use the same bottle of Vero cells to propagate eight 96-well (8 × 12-well) plates to ensure consistent cell growth. Once the cells have grown to a monolayer, proceed to the next step.

[0065] (2) Take one unthawed frozen recombinant virus of the fifth generation and the parent virus of the fifth generation, and use DNEM medium with a serum concentration of 2% for 10 -1 ~10 -10 Serial dilutions of

[0066] (3) After the dilution is completed, the original culture medium in the 96-well plate is aspirated using eight-pass pipettes in sequence. Each virus dilution is added to a column of 8 wells, with 100 μL per well. The last two columns are added with DNEM culture medium with a serum concentration of 2% as a negative control. Each virus is added to an empty 96-well plate.

[0067] (4) All 96-well plates after infection were placed in a cell culture incubator at 37°C and 5% CO2 for approximately 96 h;

[0068] (5) During the observation, the number of lesion holes at each dilution of the seed virus was recorded using a green fluorescence microscope, and the TCID of each virus was calculated using the Reed-Muench method. 50 (Table 7).

[0069] It can be clearly seen from the table that the TCID of the fifth generation virus of each recombinant virus 50 Both were lower than the fifth generation virus TCID 50 , indicating that the infectivity of each recombinant virus has decreased; among them, the recombinant viruses JS-2012-ΔgE / gI-VP1 / 2-EGFP and JS-2012-ΔgE / gI-gG-EGFP decreased the least, indicating that inserting exogenous genes at the PRV VP1 / 2 and gG gene sites has less effect on viral virulence; JS-2012-ΔgE / gI-gB-EGFP decreased the most, indicating that inserting exogenous genes at the PRV gB gene site has a greater effect on viral virulence.

[0070] Table 7 TCID of each recombinant virus and the fifth generation virus of the parent virus 50

[0071]

[0072]

[0073] After obtaining the titers of the fifth generation of each recombinant virus and parental virus, the appropriate titer was selected based on the laboratory's previous experience to inoculate cells and draw a one-step growth curve ( Figure 3 ) and compared their plaque morphology ( Figure 4 ).

[0074] The results showed that the overall proliferation rate of all recombinant viruses was lower than that of the parental virus. Among them, the overall proliferation rate of the recombinant virus JS-2012-ΔgE / gI-VP1 / 2-EGFP was closest to that of the parental virus, and the overall proliferation rate of the recombinant virus JS-2012-ΔgE / gI-TK-EGFP was the lowest. There was no significant difference in the size and morphology of the plaques between the recombinant viruses and the parental virus. Ten plaques were randomly selected from the plaque images of each recombinant virus, and the diameter of each plaque was measured and the average value was calculated. The results are shown in Figure 2. Figure 5 As shown, the results also confirmed that there was no significant difference in the plaque size of each recombinant virus.

[0075] Genetic stability analysis of recombinant viruses

[0076] The purified recombinant virus was serially passaged for 20 generations, and the fluorescence expression of each recombinant virus was observed during the passage process. The results showed that the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP showed a significant loss of fluorescence during passage, and PCR identification showed obvious spurious bands. Therefore, further passages were discontinued after the fourth passage. Figure 6 These are fluorescence images of the first and fourth generation viruses of the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP taken under a green fluorescence microscope. It can be found that obvious non-fluorescent lesions appear in the lesion area of the recombinant viruses JS-2012-△gE / gI-gC–EGFP and JS-2012-△gE / gI-UL26–EGFP, and the non-fluorescent lesions increase with the increase in the number of passages, indicating obvious loss of the target gene. Given the high difficulty in purification and the frequent difficulty in complete purification, it was concluded that these two insertion sites were unstable for exogenous gene inheritance. After passage, DNA extraction kits were used to extract DNA from 20 generations of each recombinant virus as PCR samples (DNA from F1, F10, and F20 of each recombinant virus). Parental virus DNA was also extracted as a positive control. The primers, PCR reaction system, and conditions were as in Example 1. Bacterial fluids with correct bands were sequenced by Shanghai Qingke Biotechnology Co., Ltd. and aligned with the target gene sequence using Megalign software to observe whether there were fixed gene deletions or mutations in each recombinant virus F1, F10, and F20 generations during passage. This was to observe whether the inserted target gene could be stably inherited during the 20 passages.

[0077] The results showed that the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP were completely purified and the PCR identification results of the first four generations of viral DNA were ( Figure 7 、 8 ) Based on the presence of a mixed band identical in size to the positive sample band, it was concluded that the recombinant virus had completely deleted the inserted target gene during the propagation process. This suggests that the insertion sites selected for the gC and UL26 genes are genetically unstable for foreign genes, making them difficult to propagate continuously.

[0078] Further sequencing analysis was performed on the miscellaneous bands that appeared during the PCR identification of the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP. After comparison with the reference sequence of the homology arm + target gene sequence, it was found that ( Figure 9 、 10 ): There are some base deletions and mutations in the upstream and downstream of the target gene in the hybrid band sequences of the two virus strains, and the target gene is lost as a whole, indicating that the insertion site selected on gC has poor genetic stability for exogenous genes.

[0079] The recombinant virus JS-2012-△gE / gI-gD-EGFP began to show an obvious mixed band from the 13th generation. Figure 11 As shown in the figure: With the increase of passage number, the mixed bands become more obvious. The mixed bands that appeared during the PCR identification of the recombinant virus JS-2012-△gE / gI-gD-EGFP were sequenced and analyzed. After comparison with the reference sequence of the homology arm + target gene sequence, it was found that the mixed band sequence of JS-2012-△gE / gI-gD-EGFP had a 45-base deletion upstream of the target gene compared to the expected correct band sequence, and the target gene had a 1291-base deletion ( Figure 12 ). This indicates that the insertion site selected on gD has poor genetic stability for foreign genes and can only stably inherit the target gene for about 12 generations.

[0080] The recombinant viruses JS-2012-△gE / gI-gE-EGFP, JS-2012-△gE / gI-gG-EGFP, JS-2012-△gE / gI-gB-EGFP, JS-2012-△gE / gI-gM-EGFP, JS-2012-△gE / gI-TK-EGFP and JS-2012-△gE / gI-VP1 / 2-EGFP were able to stably inherit the target gene for 20 generations ( Figure 13 ). The correct bands of the F1, F10 and F20 generations of each recombinant virus were sequenced and compared with the target gene. Figure 14As shown in the figure, the F1, F10, and F20 generations of each recombinant virus have more or less genetic mutations compared to the target gene; the genetic mutations in the F1, F10, and F20 generations of each recombinant virus do not have a fixed position, indicating that they are all random mutations. Moreover, the F1, F10, and F20 generations of each recombinant virus can express EGFP protein, indicating that these random mutations do not affect the expression of the target gene.

[0081] Vero cells were infected with the 20th generation of each recombinant virus to prepare samples for Western blot. The stability of each recombinant virus in expressing the EGFP gene was analyzed based on the results. The results are as follows: Figure 15 As shown, it was confirmed that each recombinant virus could still stably express EGFP protein at the 20th generation. ImageJ software was used to measure the grayscale values of the target protein bands and internal reference, and the relative expression levels of the EGFP proteins of each recombinant virus were calculated as shown in Table 8: the relative expression levels of the recombinant virus target proteins were arranged from large to small as JS-2012-ΔgE / gI-gE-EGFP, JS-2012-ΔgE / gI-VP1 / 2-EGFP, JS-2012-ΔgE / gI-gG-EGFP, JS-2012-ΔgE / gI-gD-EGFP, JS-2012-ΔgE / gI-gB-EGFP, JS-2012-ΔgE / gI-TK-EGFP and JS-2012-ΔgE / gI-gM-EGFP, indicating that under the same conditions, the EGFP protein expression level of JS-2012-ΔgE / gI-gE-EGFP was the highest.

[0082] Table 8 Gray values of the fifth generation virus target proteins and internal references of each recombinant virus

[0083]

[0084]

[0085] The results showed that the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP lost fluorescence at the beginning of continuous passaging, and JS-2012-△gE / gI-gD-EGFP lost fluorescence after continuous passaging to the 12th generation. PCR and sequencing verification showed that the target genes were indeed lost to varying degrees and had poor genetic stability. This shows that gC, UL26, and gD genes are not suitable as sites for exogenous gene insertion. The recombinant viruses JS-2012-△gE / gI-gE-EGFP, JS-2012-△gE / gI-gB-EGFP, JS-2012-△gE / gI-gG-EGFP, JS-2012-△gE / gI-TK-EGFP, and JS-2012-△gE / gI-gM-EGFP all exhibited good genetic stability during serial passage, and the EGFP gene was stably and efficiently expressed. However, since their cell proliferation rates were significantly lower than those of the parental virus, the gE, gB, gG, TK, and gM genes were not optimal sites for exogenous gene insertion. The recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP exhibited good genetic stability during serial passage, and the EGFP gene was stably and efficiently expressed. Its cell proliferation rate was comparable to that of the parental virus, indicating that the VP1 / 2 genes were optimal sites for exogenous gene insertion.

[0086] To further utilize the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP as a research platform, the purified virus was used for screening and preparation of monoclonal antibodies. The preparation and screening methods were described in the Molecular Cloning manual. Briefly, 6-8 week-old female BALB / c mice were immunized with the purified JS-2012-△gE / gI-VP1 / 2-EGFP virus suspension. The suspension was mixed with Freund's adjuvant (F5881-10ML) in a 1:1 ratio, fully emulsified, and then administered at multiple points on the back and intraperitoneally. Each mouse received a 200 μL booster dose. Subsequent immunizations were repeated every two weeks, with 25 μg of the virus mixed with incomplete Freund's adjuvant (F5506-10ML) in a 1:1 ratio, fully emulsified, and administered at a dose of 200 μL per mouse. Mouse antibody titers exceeding 7.2W were detected, indicating that hybridoma cell fusion was possible. After ELISA screening, positive cell lines with OD450 values greater than 0.3 were selected for subsequent expansion culture. When clones were formed, activity and subcloning were promptly tested, and the culture was gradually expanded from 96-well plates to 24-well plates and frozen in time. The hybridoma cell line with the highest titer, clone number 4H2, was selected for antibody production. 8-week-old BALB / C mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. After 1-2 weeks, the hybridoma cells were plated at 1×10 6The amount of 1 cell / mouse was injected into the abdominal cavity. 1-2 weeks after inoculation, the mouse abdomen was visibly swollen. At this time, ascites was extracted and the antibody was purified using a protein G affinity column (Huiyan Bio, HQ170827100M-100mL).

[0087] Determination of monoclonal antibody 4H2 activity using ELISA

[0088] 1) Coating Antigen: The purified recombinant virus JS-2012-ΔgE / gI-VP1 / 2-EGFP supernatant was diluted to 1 μg / mL with 1× CB buffer (1 L purified water, 33.92 g Na2CO3, 57.13 g NaHCO3, adjusted to pH 9.6, diluted 20-fold) and coated in an ELISA plate at 100 μL / well at 4°C overnight.

[0089] 2) Blocking: Wash the coated ELISA plate three times with a 1×PBST (1×PBS, 0.5% Tween-20) plate washer (300 μL / well). Pat dry, then add blocking solution (1×PBS, 1% BSA) and incubate at 37°C for 2 hours (200 μL / well). Wash the plate three times with a 1×PBST (1×PBS, 0.5% Tween-20) plate washer, pat dry, and assay.

[0090] 3) Primary Antibody: Dilute the purified antibody to 1 μg / mL in PTB diluent (1× PBS, 0.5% Tween-20, 1% BSA). Add 150 μL to well 1 of the antigen detection plate. Then perform a 1:3 serial dilution (final volume per well is 100 μL / well) for a total of 7 wells, leaving well 8 blank. Incubate at 37°C for half an hour.

[0091] 4) Secondary Antibody: Wash the plate three times with a 1× PBST plate washer (300 μL / well). Pat dry, then add 100 μL / well of a 5000-fold diluted goat anti-mouse IgG-HRP (Solarbio, Goat anti-mouse IgG, HRP-labeled, SE131-0.1ml) and incubate at 37°C for 30 minutes.

[0092] 5) Color development: Wash the plate three times with a 1× PBST plate washer, 300 μL / well. Add 100 μL / well of color development solution (Solybol, TMB two-component color development solution, PR1210-2*500ml, mix solution A and solution B in a 1:1 ratio, prepare freshly for use) and incubate at 37°C for 10 minutes.

[0093] 6) Stopping: Stopping solution 1M HCl, 50 μL / well.

[0094] 7) Data reading: OD450 was measured using a microplate reader (Radoo, RT-6100). The results are shown in Table 9, indicating that the antibody has antigen-binding activity.

[0095] Table 9 OD450 values of purified antibodies

[0096] OD450 Antibody concentration 0.615 1000 0.402 333.3 0.234 1111.1 0.167 37 0.098 12.3 0.046 4.1 0.031 1.4 0.012 comparison

[0097] The hybridoma cell line clone 4H2 was cultured to 5×10 6 The cell pellet was collected and sent to Nanjing Zhongding Biotechnology Co., Ltd. for sequencing. The amino acid sequence of the light chain variable region of the monoclonal antibody provided by the present invention is

[0098] DIVMTQSAILSSRGAKVVTMTCRASSVISSTMHWYQKPGSSAKWYATTILASGVPARFGFSGSGEASDTLTISCRVEADA ATATYCQQWQSALTFGAGAELELK (SEQ ID NO: 2), wherein CDR1-3 of the light chain variable region are RASSVISSTMH (SEQ ID NO: 4), TILAS (SEQ ID NO: 5), and QQWQSALT (SEQ ID NO: 6), respectively;

[0099] The amino acid sequence of the heavy chain variable region is

[0100] QVQLEQGPELALVKPAGSLLSCAATFTDYAMHWVRLAWEWIGYIYPYGDATHYPGYNQKFKNKATLTDNARATLYLQDLR SLTSEDATAMYYCTRDYYYGWFADVYWGTTAVTLTVSS (SEQ ID NO: 3), wherein CDR1-3 of the heavy chain variable region are MH (SEQ ID NO: 7), YIYPYGDATHYPGYNQKFKN (SEQ ID NO: 8), and DYYYGWFADVY (SEQ ID NO: 9), respectively.

[0101] In summary, this study, through systematic and extensive experimental data, confirms that the pseudorabies virus VP1 / 2 gene (32,739 bp) is the optimal site for exogenous gene insertion. Building on this foundation, we will subsequently explore inserting the E2 gene of classical swine fever virus, the VP2 gene of parvovirus, the cap gene of circovirus type 2, and the VP1 gene of foot-and-mouth disease virus, either individually or in combination, into this location to construct different types of multivalent vaccines. The successful development of such multivalent vaccines will undoubtedly revolutionize the current state of clinical swine disease vaccine use and provide advanced technical tools for swine disease prevention and control in my country.

[0102] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A recombinant pseudorabies virus strain, characterized in that: The recombinant pseudorabies virus strain is based on the pseudorabies virus variant double gene deletion vaccine strain (JS-2012-ΔgE / gI) and has exogenous genes inserted at the positions shown in Table 1. Table 1 Specific insertion sites on each gene The insertion sites are located after the CDS regions of nine genes, namely gE, VP1 / 2, gB, gC, UL26, gD, gG, TK and gM.

2. The recombinant virus according to claim 1, wherein the recombinant virus is JS-2012-ΔgE / gI-VP1 / 2-EGFP. A monoclonal antibody that can specifically recognize the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP according to claim 2, wherein the light chain variable region amino acid sequence of the monoclonal antibody is shown in SEQ ID NO: 2, the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 3, the light chain CDR1 amino acid sequence is shown in SEQ ID NO: 4, the light chain CDR2 amino acid sequence is shown in SEQ ID NO: 5, the light chain CDR3 amino acid sequence is shown in SEQ ID NO: 6, the heavy chain CDR1 amino acid sequence is shown in SEQ ID NO: 7, the heavy chain CDR2 amino acid sequence is shown in SEQ ID NO: 8, and the heavy chain CDR3 amino acid sequence is shown in SEQ ID NO:

9.

3. A recombinant pseudorabies virus vaccine construction platform, characterized in that The platform is the JS-2012-ΔgE / gI-VP1 / 2-EGFP described in claim 2.

4. The platform according to claim 4, characterized in that The E2 gene of classical swine fever virus, the VP2 gene of parvovirus, the cap gene of circovirus type 2, and the VP1 gene of foot-and-mouth disease virus are replaced or inserted into the EGFP expression frame individually or in combination to construct different types of multi-component vaccines.

5. Use of the recombinant pseudorabies virus vaccine construction platform according to claim 4 or 5 in the preparation of a vaccine.

6. Use of the recombinant virus JS-2012-ΔgE / gI-VP1 / 2-EGFP according to claim 2, the monoclonal antibody according to claim 3, and / or the platform according to claim 4 in the preparation of a medicament for treating or preventing a disease caused by pseudorabies virus.

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