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

Abstract

The application provides a construction and application of a recombinant pseudorabies virus strain stably and efficiently expressing green fluorescent protein, in the research, a green fluorescent protein gene (EGFP) is inserted into different positions of a double gene deletion vaccine strain (JS-2012-△gE / gI) of a pseudorabies virus mutant strain by using a homologous recombination method. Finally, it is proved that the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP has good genetic stability, and the EGFP gene can also be stably and efficiently expressed, and the growth rate on cells is also comparable to that of the parent virus, so that the VP1 / 2 gene is the best exogenous gene insertion site. Subsequently, 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 replaced or inserted into the EGFP expression frame in a single or combined manner to construct different types of multi-vaccines, and the multi-vaccines have extremely high research value and platform significance.

Description

Technical Field

[0001] This invention belongs to the field of animal medicine, specifically involving the basic research and development of a porcine pseudorabies virus gene-deleted vaccine strain, and particularly the construction and application of a recombinant pseudorabies virus strain that stably and efficiently expresses green fluorescent protein. Background Technology

[0002] Pseudorabies virus (PRV) belongs to the alphaherpesvirus subfamily of the herpesviridae family. It is a double-stranded DNA virus that infects pigs and causes porcine pseudorabies (Aujeszky's disease), causing significant economic losses to the global pig industry. Due to its genome's ability to accommodate a large number of exogenous genes and its broad host range, PRV has become an important tool for research on live viral vector vaccines and neural circuit tracing, thanks to advancements in genetic engineering technology. The selection of different insertion sites is a key factor in the success or failure of live viral vector vaccine development. Existing studies have shown that replication-non-essential regions of the PRV genome (such as the TK gene and gE / gI gene regions) are often chosen as insertion sites for exogenous genes. However, recombinant viruses with different insertion sites exhibit differences in replication efficiency, genetic stability, and exogenous gene expression levels in cells. Furthermore, the selection of insertion sites must consider both the expression regulation of exogenous genes and the synergy with the virus's own genes. However, a systematic study on different insertion sites in the PRV genome is still lacking.

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

[0004] To address the aforementioned issues, this application first provides a set of recombinant viruses based on different exogenous genes of the parental virus JS-2012-ΔgE / gI. The specific insertion sites of these recombinant viruses are shown in Table 1.

[0005] Table 1. Specific insertion sites on each gene.

[0006]

[0007]

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

[0009] The nine recombinant viruses obtained were 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, this application provides a recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP, which exhibits good genetic stability during continuous passage and the EGFP gene can also be expressed stably and efficiently. Its proliferation rate on cells is also comparable to that of the parent virus. Therefore, the VP1 / 2 gene is the best exogenous gene insertion site.

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

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

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

[0014] Furthermore, the present invention provides the use of recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP, monoclonal antibody, and vaccine construction platform in the preparation of drugs 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 in the pseudorabies virus variant double-gene deletion vaccine strain (JS-2012-△gE / gI) using homologous recombination. The insertion sites were located after the CDS regions of nine genes: gE, VP1 / 2, gB, gC, UL26, gD, gG, TK, and gM. 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. The nine recombinant viruses were blindly passaged for 20 generations in Vero cells to verify their genetic stability. The replication ability, EGFP protein expression level, and other biological characteristics of the fifth-generation recombinant viruses were compared and analyzed. The results showed that the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP exhibited fluorescence loss at the beginning of continuous passaging, while JS-2012-△gE / gI-gD-EGFP showed fluorescence loss after 12 consecutive passaging. Subsequent PCR and sequencing confirmed that the target genes did indeed have varying degrees of loss and poor genetic stability. Therefore, the gC, UL26, and gD genes are not suitable as exogenous gene insertion sites. 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 continuous passaging, and the EGFP gene was consistently and efficiently expressed. However, because their cell proliferation rates were significantly lower than those of the parent virus, the gE, gB, gG, TK, and gM genes were not optimal insertion sites for foreign genes. The recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP also exhibited good genetic stability during continuous passaging, and the EGFP gene was consistently and efficiently expressed. Its cell proliferation rate was comparable to that of the parent virus; therefore, the VP1 / 2 gene was the optimal insertion site for foreign genes.

[0017] This study, by constructing multiple recombinant viruses with different insertion sites and combining them with in vitro cell experiments, systematically revealed the mechanism by which different sites affect viral phenotypes. This research is expected to fill a gap in this field and deepen our understanding of the modular functional characteristics of the PRV genome. From an application perspective, screening for efficient and safe insertion sites directly promotes PRV vaccine development. First, optimizing insertion sites can improve the safety of gene-deleted vaccines. Second, the choice of insertion site may affect the expression level of exogenous antigens and the strength of the immune response. For example, certain gene regions (such as downstream of the promoter of early-expressing genes) may be more conducive to the efficient transcription of exogenous genes, thereby enhancing the multivalent immunization effect 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 screening for genetically stable sites, the risk of gene rearrangement or loss during the passage of recombinant viruses can be reduced, providing technical support for PRV as a vaccine vector (such as for delivering African swine fever virus antigens). Further, this research has significant implications for the sustainable development of animal husbandry. Pseudorabies control is a core aspect of swine herd health management, and the optimization of genetically engineered vaccines can reduce latent infection or virulence reversion problems that may be caused by traditional vaccines (such as attenuated live vaccines). By screening for the optimal insertion site, the development of next-generation marker vaccines (DIVA vaccines) can be promoted, achieving an efficient combination of disease eradication and surveillance. In addition, as a model pathogen of neurotropic viruses, the optimization of PRV vector systems can also provide cross-species references for gene therapy research on human herpesviruses (such as HSV-1), and has potential translational medicine value. Attached Figure Description

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

[0019] Figure 2 Identification results of purified co-transfected viruses;

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

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

[0022] Figure 5 Statistical analysis of plaque diameters for each recombinant virus and parent virus;

[0023] Figure 6 Fluorescence images of JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP during passage.

[0024] Figure 7PCR identification of JS-2012-△gE / gI-gC-EGFP co-transfected virus and its 4th generation;

[0025] Figure 8 PCR identification of JS-2012-△gE / gI-UL26-EGFP co-transfected virus and its 4th generation;

[0026] Figure 9 Sequencing analysis of JS-2012-△gE / gI-gC-EGFP PCR heterogeneous bands;

[0027] Figure 10 JS-2012-△gE / gI-UL26-EGFP PCR heterogeneous band sequencing analysis;

[0028] Figure 11 JS-2012-△gE / gI-UL26-EGFP PCR heterogeneous band sequencing analysis, where M2: Trans 2K DNA Marker; 1-20: 1st-20th generation recombinant virus samples; 21: positive control; 22: negative control;

[0029] Figure 12 Sequencing analysis of JS-2012-△gE / gI-gD-EGFP PCR heterogeneous bands;

[0030] Figure 13 3-11 PCR identification of each recombinant virus at passage 20: M1: Trans 5K DNA Marker; M2: Trans 2K DNA Marker; 1-20: Recombinant virus samples at passages 1-20; 21: Positive control; 22: Negative control;

[0031] Figure 14 The results of sequencing of the genes of each recombinant virus generation FI, F10, and F20 and the alignment of the target gene;

[0032] Figure 15Western blot results of EGFP protein expression in the 20th generation of various recombinant viruses. M: protein molecular weight standard; 1: Western blot results of EGFP protein expression in JS-2012-ΔgE / gI-VP1 / 2-EGFP; 2: Western blot results of EGFP protein expression in JS-2012-ΔgE / gI-gE-EGFP; 3: Western blot results of EGFP protein expression in JS-2012-ΔgE / gI-gD-EGFP; 4: Western blot results of EGFP protein expression in JS-2012-ΔgE / gI-gG-EGFP; 5: Western blot results of EGFP protein expression in JS-2012-ΔgE / gI-gB-EGFP; 6: Western blot results of EGFP protein expression in JS-2012-ΔgE / gI-TK-EGFP. blot results; 7: Western blot results of EGFP protein expression of JS-2012-ΔgE / gI-gM-EGFP; 8: Positive control JS-2012-ΔgE / gI; 9: Negative control. Detailed Implementation

[0033] The cell viruses used included 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, Hao Zheng, 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 were kept in our laboratory. Other reagents, instruments, and equipment were standard procedures in the field and selected according to specific circumstances, and are not considered as the inventive point of this application.

[0034] Example 1: Determination of the specific insertion site of the recombinant pseudorabies virus strain

[0035] Based on the applicant's preliminary research, the final selection of insertion sites was made for the gE, VP1 / 2, gD, gG, gB, TK, gM, gC, and UL26 genes of the parental virus JS-2012-ΔgE / gI. The specific insertion sites selected in the parental viral gene sequences are shown in Table 1.

[0036] Table 1. Specific insertion sites on each gene.

[0037]

[0038] Based on the existing PRV mutation deletion strain JS-2012-ΔgE / gI full genome sequence in our laboratory, homologous arms were designed upstream and downstream of each insertion site using Primer 5.0. The cloning vector chosen was pBluescript ll SK(+), and the target gene was the CMV+EGFP+SV40 sequence, both of which were preserved in our 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 preserved in our laboratory. The construction of transfer vectors corresponding to the insertion sites on the VP1 / 2, gD, gB, TK, gM, gC, and UL26 genes was completed by Shanghai Saiheng Biotechnology Co., Ltd., and named 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, respectively. All of the above vectors were sequenced to verify their correctness, and the nucleotide sequence of pBluescript ll SK(+)-VP1 / 2-EGFP is shown in SEQ ID NO.1.

[0039] The specific left and right homologous arm regions on each transfer vector are shown in Table 2:

[0040] Table 2 shows the specific left and right homologous arm regions on each transfer vector.

[0041]

[0042]

[0043] Sequencing confirmed the successful preparation of the above-mentioned transfer vector plasmids. After large-scale culture, the plasmids were transfected into BHK cells to observe the expression levels of each transfer vector and verify their activity. 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 transfected vector. Results are as follows: Figure 1 As shown, this confirms that by constructing specific eukaryotic expression vectors and transforming them into cells, each transfer vector can be efficiently expressed in cells.

[0044] To improve the transfection efficiency of co-transfection, the transfer vectors were linearized. The transfer vectors pBluescript llSK(+)-gE-EGFP and pBluescript ll SK(+)-gG-EGFP were linearized using XhoI restriction endonuclease, while the transfer vectors 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 were linearized using HindIII-HF restriction endonuclease. Simultaneously, the genome of the JS-2012-△gE / gI strain was extracted using the phenol-chloroform method.

[0045] 2 μg of linearized transfer vector and 2 μg of parental viral genome were co-transfected. After co-transfecting, the recombinant viruses were purified using Vero cells. Once all recombinant viruses were completely purified, they were identified by PCR.

[0046] Based on the left and right homologous 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 were all stored in the laboratory. Specific primer names and sequences are shown in Table 3 below:

[0047] Table 3. Names and sequences of primers for identifying recombinant viruses.

[0048]

[0049] Based on the identification results, the PCR reaction identification methods for various recombinant viruses are divided into two types:

[0050] The recombinant viruses JS-2012-△gE / gI-gE-EGFP, JS-2012-△gE / gI-gB-EGFP, JS-2012-△gE / gI-gC-EGFP, JS-2012-△gE / gI-VP1 / 2-EGFP, and JS-2012-△gE / 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.

[0051] Table 4. Identification system for LA Taq enzyme

[0052]

[0053] Recombinant viruses JS-2012-△gE / gI-gD-EGFP, JS-2012-△gE / gI-TK-EGFP, JS-2012-△gE / gI-gG-EGFPH, and JS-2012-△gE / gI-gM-EGFP were identified using 2×GC-rich Master Mix. The reaction volume was 20 μL, and the amounts of each component are shown in Table 5.

[0054] Table 5 Identification System of 2×GC-rich Master Mix

[0055]

[0056] The PCR reaction conditions for the identification of each recombinant virus differed only in annealing temperature and extension time, as shown in Table 6. The other reaction conditions were consistent, in the following order: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, annealing for 30 s, 72℃ extension, and 72℃ final extension for 10 min, for a total of 35 reaction cycles.

[0057] Table 6 Annealing temperature and extension time for PCR identification of various recombinant viruses.

[0058]

[0059] The results are as follows Figure 2 As shown, all the identified bands matched the expectations, indicating that pure recombinant viruses were obtained.

[0060] Example 2: Biological Characteristics Analysis of Recombinant Virus

[0061] Virus growth characteristics analysis

[0062] Fifth-generation recombinant viruses and fifth-generation parental viruses were selected for titer determination and comparison. The specific procedures are as follows:

[0063] (1) Use the same bottle of Vero cells to transfer to 8 96 (8×12) well plates to ensure that the cells grow in the same state. Once the cells have grown to a monolayer, prepare to proceed to the next step.

[0064] (2) Take one unthawed frozen recombinant fifth-generation virus and one parental fifth-generation virus, and sequentially perform 10 tests on DNEM medium with a serum concentration of 2%. -1 ~10 -10 Gradient dilution;

[0065] (3) After dilution, the original culture medium in the 96-well plate was aspirated sequentially using an eight-channel pipette. Each dilution of virus was added to one column of 8 wells, 100 μL per well. The last two columns were added with 2% DNEM culture medium as a negative control. Each type of virus was added to one 96-well empty plate.

[0066] (4) Place all the 96-well plates after inoculation in a cell culture incubator at 37°C with a CO2 concentration of 5% for about 96 hours;

[0067] (5) During the period, the number of lesion wells of each virus at each dilution was observed and recorded using a green fluorescence microscope. Finally, the TCID of each virus was calculated using the Reed-Muench method. 50 (Table 7)

[0068] The table clearly shows the TCID of each recombinant fifth-generation virus. 50 All were lower than the TCID of the fifth-generation parent virus. 50 This indicates 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 showed the smallest decrease, indicating that the insertion of foreign genes at the PRV VP1 / 2 and gG gene sites has a smaller impact on viral virulence; JS-2012-ΔgE / gI-gB-EGFP showed the largest decrease, indicating that the insertion of foreign genes at the PRV gB gene site has a larger impact on viral virulence.

[0069] Table 7. TCID of each recombinant virus and its parental fifth-generation virus 50

[0070]

[0071] After obtaining the viral titers of each recombinant virus and the fifth-generation parental virus, appropriate viral titers were selected based on previous laboratory experience for cell inoculation, and a one-step growth curve was plotted. Figure 3 And compare their morphology of empty spots ( Figure 4 ).

[0072] The results showed that the overall proliferation rate of all recombinant viruses was lower than that of the parent viruses. Among them, the overall proliferation rate of recombinant virus JS-2012-ΔgE / gI-VP1 / 2-EGFP was closest to that of the parent virus, while the overall proliferation rate of recombinant virus JS-2012-ΔgE / gI-TK-EGFP was the lowest. There were no significant differences in plaque size and morphology between the recombinant viruses and the parent viruses. Ten plaques were randomly selected from the plaque maps of each recombinant virus, and the diameter of each plaque was measured and the average value was calculated. The results are shown below. Figure 5 As shown, the results also confirm that there is no significant difference in the size of the plaques among the recombinant viruses.

[0073] Genetic stability analysis of recombinant viruses

[0074] The fully purified recombinant virus was passaged 20 times consecutively, and the fluorescence expression of each recombinant virus at each passage 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 exhibited significant fluorescence loss during passage, and PCR identification revealed obvious heterogeneous bands. Therefore, passage was discontinued after the fourth passage. Figure 6 The images show fluorescence images of the first and fourth generation recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP, taken under a green fluorescence microscope. It can be observed that the lesion areas of each generation of JS-2012-△gE / gI-gC–EGFP and JS-2012-△gE / gI-UL26–EGFP show obvious non-fluorescent lesions, and the number of non-fluorescent lesions increases with each generation, indicating a significant loss of the target gene. Given the difficulty in purification and the frequent incomplete purification, it was determined that these two insertion sites are unstable for the inheritance of exogenous genes. After passage, DNA was extracted from 20 generations of each recombinant virus using a DNA extraction kit as PCR samples (DNA from F1, F10, and F20 of each recombinant virus). Parental viral DNA was also extracted as a positive control. Primers, PCR reaction system, and conditions were as described in Example 1. Bacterial cultures with correct bands were sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing. The sequences were compared with the target gene sequence using Megalign software to observe whether there were fixed gene deletions or mutations in the F1, F10, and F20 generations of each recombinant virus during passage. This was to observe whether the inserted target gene could be stably inherited during 20 generations of passage.

[0075] The results showed that the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP, after complete co-transfection and PCR identification of the first four generations of viral DNA, were effective. Figure 7 , 8 Based on the presence of heterogeneous bands of the same size as those in the positive samples during identification, it can be concluded that the target gene inserted during the propagation process of the recombinant virus was completely deleted. Therefore, it is inferred that the insertion sites selected in the gC and UL26 genes have poor genetic stability for the exogenous gene, making continuous passaging difficult.

[0076] Further analysis of the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△

[0077] Sequencing analysis was performed on the extraneous bands that appeared during the gE / gI-UL26-EGFP PCR identification process. After alignment with the reference sequence of the homologous arm + target gene sequence, it was found that... Figure 9 , 10 The two viral strains showed some base deletions and mutations upstream and downstream of the target gene in their hybrid sequences, and the target gene was completely lost, indicating that the selected insertion site on gC has poor genetic stability for exogenous genes.

[0078] The recombinant virus JS-2012-△gE / gI-gD-EGFP showed a distinct heterogeneous band starting from generation 13. The results are as follows... Figure 11 As shown, the heterogeneous bands became increasingly prominent with each passage. Sequencing analysis of the heterogeneous bands appearing during PCR identification of the recombinant virus JS-2012-△gE / gI-gD-EGFP revealed that, compared to the expected correct band sequence, the heterogeneous band sequence of JS-2012-△gE / gI-gD-EGFP had a 45-base deletion upstream of the target gene, and the target gene itself had a 1291-base deletion. Figure 12 This indicates that the insertion site selected on gD has poor genetic stability for exogenous genes, and can only stably transmit the target gene for about 12 generations.

[0079] 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 can all stably transmit 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 the alignment results with the target gene are as follows: Figure 14As shown, the F1, F10, and F20 generations of each recombinant virus have varying degrees of gene mutations compared to the target gene. The gene mutations in the F1, F10, and F20 generations of each recombinant virus do not have fixed locations, indicating that they are all random mutations. Furthermore, 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.

[0080] Vero cells were infected with the 20th generation of each recombinant virus to prepare samples, which were then subjected to Western blot analysis. The stability of each recombinant virus in EGFP gene expression was analyzed based on the results. Figure 15 As shown, it is confirmed that each recombinant virus can still stably express EGFP protein in the 20th generation. The target protein bands and internal controls were measured using ImageJ software to determine their grayscale values. The relative expression levels of EGFP proteins from each recombinant virus were calculated, as shown in Table 8. The relative expression levels of the target proteins from each recombinant virus, arranged from highest to lowest, are: 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. This indicates that under the same conditions, JS-2012-ΔgE / gI-gE-EGFP exhibits the highest EGFP protein expression level.

[0081] Table 8. Gray values ​​of target proteins and internal controls for each fifth-generation recombinant virus.

[0082]

[0083]

[0084] The results showed that the recombinant viruses JS-2012-△gE / gI-gC-EGFP and JS-2012-△gE / gI-UL26-EGFP exhibited fluorescence loss at the beginning of continuous passaging, while JS-2012-△gE / gI-gD-EGFP showed fluorescence loss after 12 consecutive passaging. Subsequent PCR and sequencing confirmed that the target genes did indeed have varying degrees of loss and poor genetic stability. Therefore, the gC, UL26, and gD genes are not suitable as exogenous gene insertion sites. 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 continuous passaging, and the EGFP gene was consistently and efficiently expressed. However, because their cell proliferation rates were significantly lower than those of the parent virus, the gE, gB, gG, TK, and gM genes were not optimal insertion sites for foreign genes. The recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP also exhibited good genetic stability during continuous passaging, and the EGFP gene was consistently and efficiently expressed. Its cell proliferation rate was comparable to that of the parent virus; therefore, the VP1 / 2 gene was the optimal insertion site for foreign genes.

[0085] To further utilize the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP as a research platform, monoclonal antibodies were screened and prepared using the purified virus described above. The preparation and screening methods were performed according to the "Molecular Cloning" manual. In summary: 6-8 week old female BALB / c mice were initially immunized with the purified JS-2012-△gE / gI-VP1 / 2-EGFP virus collection solution, which was then thoroughly emulsified with Freund's adjuvant (F5881-10ML) at a 1:1 ratio. The mixture was then immunized at multiple sites on the back and intraperitoneally, with a dose of 200 μL per mouse. Subsequent booster immunizations were performed every two weeks, with each booster immunization consisting of 25 μg of the virus mixed with Freund's incomplete adjuvant (F5506-10ML) at a 1:1 ratio and thoroughly emulsified, with a dose of 200 μL per mouse. When the antibody titer in the mice exceeded 7.2W, subsequent hybridoma cell fusion was considered feasible. After ELISA screening, positive cell lines with OD450 values ​​greater than 0.3 were selected for subsequent expansion culture. When clonal clusters formed, cell viability and subclones were promptly detected. Cells were progressively expanded from 96-well plates to 24-well plates and cryopreserved. The hybridoma cell line with the highest titer, clone number 4H2, was selected for antibody production. 0.5 mL of Freund's incomplete adjuvant was injected intraperitoneally into 8-week-old BALB / c mice. One to two weeks later, hybridoma cells were cultured at a rate of 1 × 10⁻⁶ cells / mL. 6The amount of cells per mouse is injected into the peritoneal cavity. One to two weeks after inoculation, the abdomen of the mouse will be distended. At this time, the ascites can be aspirated and the antibody can be purified using a protein G affinity column (Huiyan Biotechnology, HQ170827100M-100mL).

[0086] Determining the activity of monoclonal antibody 4H2 using ELISA

[0087] 1) Coating antigen: The purified recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP virus supernatant was diluted to 1 μg / mL with 1×CB buffer (1L purified water with 33.92g Na2CO3 and 57.13g NaHCO3 added, pH adjusted to 9.6, diluted 20 times), and coated onto ELISA microplates, 100 μL / well, and incubated overnight at 4℃.

[0088] 2) Blocking: Wash the coated microplate three times with 1×PBST (1×PBS, 0.5% Tween-20), 300 μL / well. After drying, add blocking buffer (1×PBS, 1% BSA) and incubate at 37°C for 2 hours, 200 μL / well. Wash three times with 1×PBST (1×PBS, 0.5% Tween-20), and dry before assay.

[0089] 3) Primary antibody: Dilute the purified antibody to 1 μg / mL using PTB dilution buffer (1×PBS, 0.5% Tween-20, 1% BSA). Add 150 μL to well #1 (the first well) of the antigen detection strip, and then perform a 1:3 longitudinal serial dilution (the final volume after serial dilution is 100 μL / well), for a total of 7 wells. The 8th well is a blank. Incubate at 37°C for half an hour.

[0090] 4) Secondary antibody: Wash the plate 3 times with a 1×PBST washer, 300 μL / well. After patting dry, add 100 μL / well of 5000-fold diluted goat anti-mouse IgG-HRP (Solepro, Goat anti-mouse IgG, HRP-labeled, SE131-0.1 ml) and incubate at 37°C for half an hour.

[0091] 5) Color development: Wash the plate 3 times with a 1×PBST washer, 300 μL / well. Add 100 μL / well of color development solution (Soleb, TMB two-component color development solution, PR1210-2*500ml, A and B solutions mixed in a 1:1 ratio, freshly prepared and used immediately), and develop at 37℃ for 10 minutes.

[0092] 6) Termination: 1M HCl, 50μL / well.

[0093] 7) Data reading: Using an ELISA reader (Redu, RT-6100), the OD450 was measured. The results are shown in Table 9, indicating that the antibody has antigen-binding activity.

[0094] Table 9. OD450 values ​​of purified antibodies

[0095] 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

[0096] Hybridoma cell line 4H2 was cultured to 5 × 10⁶ cells / year. 6 Cells were collected, and the cell pellet was 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 this invention is DIVMTQSAILSSRGAKVVTMTCRASSVISSTMHWYQKPGSSAKWYATTILASGVPARFGFSGSGEASDT LTISCRVEADAATATYCQQWQSALTFGAGAELELK (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.

[0097] The amino acid sequence of the heavy chain variable region is as follows

[0098] QVQLEQGPELALVKPAGSLLSCAATFTDYAMHWVRLAWEWIGYIYPYGDATHYPGYNQKFKNKATLTDNARATLYLQDLRSLTSEDATAMYYCTRDYYYGWFADVYWGTTAVTLTVSS (SEQ ID NO:3), wherein the 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.

[0099] In summary, this study, through systematic and extensive experimental data, has confirmed that the VP1 / 2 gene (32739 bp) of pseudorabies virus is the optimal insertion site for exogenous genes. Going forward, we will also explore inserting the classical classical swine fever virus E2 gene, parvovirus VP2 gene, porcine circovirus type 2 cap gene, and foot-and-mouth disease virus VP1 gene, individually or in combination, into this site to construct different types of multivalent vaccines. If such multivalent vaccines can be successfully developed, it will undoubtedly revolutionize the current state of clinical swine disease vaccine use and provide advanced technical means for swine disease prevention and control in my country.

[0100] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A recombinant pseudorabies virus strain, characterized in that, The recombinant pseudorabies virus strain used as the starting strain was a pseudorabies virus variant strain with double gene deletion, namely JS-2012-△gE / gI. There was an insertion of a foreign gene after the CDS region of the VP1 / 2 gene, specifically at the insertion site of 32740 bp.

2. The recombinant virus as described in claim 1, wherein the recombinant virus is JS-2012-△gE / gI-VP1 / 2-EGFP.

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

4. The platform as described in claim 3, characterized in that... Different types of multivalent vaccines can be constructed by replacing or inserting the classical swine fever virus E2 gene, parvovirus VP2 gene, porcine circovirus type 2 cap gene, and foot-and-mouth disease virus VP1 / 2 gene individually or in combination into the EGFP expression cassette.

5. The application of the recombinant pseudorabies virus vaccine construction platform as described in claim 3 or 4 in vaccine preparation.

6. Use of the recombinant virus of claim 2 and / or the platform of claim 3 in the preparation of a medicament for treating or preventing diseases caused by pseudorabies virus.

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