Duck plague virus LORF2 gene deleted strain as well as construction method and application thereof

Abstract

The invention discloses a duck plague virus LORF2 gene deleted strain as well as a construction method and application thereof, and belongs to the technical field of gene engineering. The construction method comprises the following steps: S1, by taking a pEP-Kan-S plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification on a delta LORF2-Kan targeting fragment; s2, electrically transferring the targeting fragment to a competent state to construct a positive bacterium of which the LORF2 gene is replaced by a Kan resistance gene; s3, knockout of Kan resistance genes through homologous recombination; and S4, carrying out virus rescue to obtain the LORF2 gene deleted strain. The duck plague virus LORF2 gene deletion strain constructed by the invention has reduced pathogenicity to ducks and high immune factor level, can completely protect the ducks from being attacked by virulent viruses by immunizing the ducks with an ultra-low dose of 102 TCID50, has a virulent virus removal effect and a neutralizing antibody level which are similar to those of an existing commercial vaccine group, and provides strains, thoughts and technical supports for construction of DPV attenuated live vaccines.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a duck plague virus LORF2 gene deletion strain, its construction method, and its application. Background Technology

[0002] Duck plague (DP), also known as duck viral enteritis (DVE), is a highly contagious acute septicemic disease caused by the duck plague virus (DPV). This disease primarily affects anseriformes birds such as ducks and geese, and is characterized by rapid spread, wide distribution, acute onset, and high mortality. It has become one of the most destructive infectious diseases facing the global duck farming industry, causing significant economic losses.

[0003] DPV is a spherical virus with an enveloped structure, and its viral particle diameter is approximately 160-180 nm. This virus belongs to the Herpesviridae family (…). Herpesviridae Marek's virus genus ( Mardivirus Avian herpesviruses (DPVs) are double-stranded DNA viruses. Genes specific to avian herpesviruses are those that are not homologous to other herpesviruses and constitute only a small portion of the entire genome. They are usually named using the form ORF followed by a number, or according to their location: LORF for those located in the UL region, RLORF for those in inverted repeat long (IRL) regions, SORF for those in the US region, and RSORF for those in inverted repeat short (IRS) regions. Five such genes have been identified in DPVs: four located in the UL region (LORF2 (vLIP), LORF3, LORF4 (LORF9), and LORF5 (LORF11), and one located in the US region (SORF3). The functions of these genes and their encoded proteins are largely unknown, and their impact on viral pathogenicity is also largely unknown.

[0004] The LORF2 gene has been reported in MDV, HVT, DPV, and FaHV-1, and its homologs exist in some avian adenoviruses. Amino acid homology analysis shows that the proteins encoded by the LORF2 genes in DPV, MDV, and FaHV-1 contain a highly conserved functional domain related to the pancreatic lipase family catalytic triplet. This domain is a typical catalytic center formed by a hydrogen bond network of three key amino acid residues: serine (Ser), aspartic acid (Asp), and histidine (His). However, in MDV, although the lipase-catalyzing triplet domain of the LORF2 protein retains several residues crucial for lipase activity, the lipase catalytic activity of the MDV LORF2 protein is lost due to the substitution of aspartic acid by asparagine. Furthermore, the mutation of serine to alanine in the triplet severely affects MDV virulence. Studies have also found that promoter or intron mutations in LORF2 can reduce the lethality of MDV in chickens.

[0005] The LORF2 gene, a gene specific to avian herpesviruses, has only been reported to reduce viral virulence in Marek's disease virus (MDV) strains, but the reason for this reduction in virulence remains unknown. Furthermore, the function of the LORF2 gene and its encoded protein in duck plague (DPV), as well as its impact on DPV virulence, is unclear. Whether it is possible to construct a basic strain for the immunoprotection of duck plague through research on the LORF2 gene is still uncertain. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a duck plague virus LORF2 gene deletion strain, its construction method, and its application.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a method for constructing a duck plague virus LORF2 gene deletion strain, comprising the following steps: S1: Target fragment amplification Using pEP-Kan-S plasmid as a template, PCR amplification was performed using primers ΔLORF2-Kan-F and ΔLORF2-Kan-R as shown in SEQ ID NO.1~2 to obtain the ΔLORF2-Kan targeting fragment; S2: Homologous recombination to knock out the LORF2 gene The ΔLORF2-Kan targeting fragment was electroporated into pDPV-CHv50 (GS1783) competent cells. After antibiotic screening and PCR identification, positive bacteria pDPV-ΔLORF2-Kan with the Kan resistance gene replaced by the LORF2 gene (i.e., the LORF2 gene is missing) were obtained. S3: Homologous recombination to knock out the Kan resistance gene pDPV-ΔLORF2-Kan was induced and cultured, the Kan resistance gene was knocked out and PCR identification was performed to obtain the infectious clone pDPV-ΔLORF2 with the LORF2 gene missing; S4: Virus Rescue Infective clone pDPV-ΔLORF2 plasmid was extracted and transfected into DEF cells to obtain the LORF2 gene deletion strain DPV-ΔLORF2.

[0008] Furthermore, the PCR amplification system in S1 was a 50 µL system, including: 25 µL of 2× high-fidelity enzyme premix, 1 µL of template pEP-Kan-S plasmid, 1 µL each of ΔLORF2-Kan-F / R, and 22 µL of ddH2O; the PCR amplification program was: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 30 cycles, and a final extension at 72℃ for 10 min.

[0009] Furthermore, the primers used for PCR identification in S2 are LORF2-R as shown in SEQ ID NO.4 and Kan-R as shown in SEQ ID NO.6, or LORF2-JD-F / R as shown in SEQ ID NO.7~8.

[0010] Furthermore, the primers used for PCR identification in S3 are LORF2-F / R as shown in SEQ ID NO.3~4.

[0011] In a second aspect, the present invention provides a duck plague virus LORF2 gene deletion strain, which is prepared by the above-described construction method.

[0012] A third aspect of the present invention provides a method for constructing a duck plague virus LORF2 gene revertant strain, comprising the following steps: (1) Target fragment amplification: Using pDPV-CHv50 plasmid as a template and LORF2-F / R as primers, the LORF2 homologous fragment was amplified; using pEPKan-S plasmid as a template and Kan-F / R as primers, the Kan homologous fragment was amplified; using the LORF2 homologous fragment and the Kan homologous fragment as templates, and using LORF2-F and Kan-R as primers, fusion PCR was performed to obtain the targeting fragment LORF2-Kan; (2) Homologous recombination reverts to the LORF2 gene The targeting fragment LORF2-Kan was electroporated into the competent cells of the infectious clone pDPV-ΔLORF2 obtained in claim 1. After antibiotic screening and PCR identification, a positive bacterium pDPV-ΔLORF2-Rev-Kan containing the LORF2 gene reversion and the Kan resistance gene was obtained. (3) Homologous recombination knockout of Kan resistance gene The positive bacteria pDPV-ΔLORF2-Rev-Kan obtained from S2 were induced and cultured, the Kan resistance gene was knocked out, and PCR identification was performed to obtain an infectious clone pDPV-ΔLORF2-Rev with LORF2 gene recovery. (4) Virus rescue The infectious clone pDPV-ΔLORF2-Rev plasmid was extracted and transfected into DEF cells to obtain the LORF2 gene deletion revertant strain DPV-ΔLORF2-Rev.

[0013] Further, in step (1), the sequences of the LORF2-F / R primers are shown in SEQ ID NO.3~4; the sequences of the Kan- / R primers are shown in SEQ ID NO.5~6.

[0014] Furthermore, the PCR amplification systems for amplifying the LORF2 homologous fragment and the Kan homologous fragment in step (1) were as follows: 0.5 μL template, 5 μL PrimerStar Max DNA Polymerase, 0.3 μL each of upstream and downstream primers, and ddH2O added to 10 μL; the PCR amplification program was as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 30 s, for a total of 30 cycles, and finally 72℃ extension for 10 min.

[0015] Further, the fusion PCR system in step (1) is as follows: PrimerStar Max DNA Polymerase 5 μL, LORF2-F 0.5 μL, Kan-R 0.5 μL, LORF2 homologous fragment and Kan homologous fragment 0.5 μL each, and ddH2O added to 10 μL; the fusion PCR program is as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 2 min, for a total of 30 cycles, and finally 72℃ extension for 10 min.

[0016] Furthermore, the primers used for PCR identification in step (2) are LORF2-F / R as shown in SEQ ID NO.3~4.

[0017] Furthermore, the primers used for PCR identification in step (3) are LORF2-JD-F / R, such as SEQ ID NO.7~8.

[0018] In a fourth aspect, the present invention provides the application of the above-mentioned duck plague virus LORF2 gene deletion strain in the preparation of duck plague virus vaccine.

[0019] The present invention has the following beneficial effects: This invention provides a method for constructing a duck plague virus (DPV) strain with the LORF2 gene deletion, resulting in the DPV-ΔLORF2 strain. Experiments demonstrate that this invention reduces the pathogenicity of DPV to ducks by deleting the LORF2 gene, while simultaneously increasing the levels of immune factors in ducks. Even at 10... 2 TCID 50 The ultra-low dose of immunization can completely protect ducks from the attack of the virulent virus, and has a clearance effect and neutralizing antibody level similar to existing commercial vaccine groups, providing sufficient technical support for the construction of a live attenuated vaccine for duck plague virus. Attached Figure Description

[0020] Figure 1 This describes the construction principle and process of pDPV-ΔLORF2 and pDPV-ΔLORF2-Rev in Example 1, where A is pDPV-ΔLORF2 and B is pDPV-ΔLORF2-Rev. Figure 2 The results of constructing DPV-ΔLORF2 in Example 1 are shown below. A represents the colony PCR identification results of the target knockout of the LORF2 gene, B represents the PCR identification results of pDPV-ΔLORF2-Kan homologous recombination after removing the Kan resistance gene, C represents the fluorescence characterization results of pDPV-ΔLORF2 transfected into DEF cells, and D represents the fluorescence characterization results of purified DPV-ΔLORF2 strain with the DPV LORF2 gene deletion. Figure 3 The images show the construction results of DPV-ΔLORF2Rev in Example 1. A represents the agarose gel electrophoresis images of the LORF2 homologous fragment, the Kan homologous fragment, and their fusion fragment; B represents the PCR identification results of the positive clone that reverted to the LORF2 gene after targeting; C represents the colony PCR identification results of the infectious clone pDPV-ΔLORF2Rev, a duck plague virus LORF2 gene deletion revertant strain after homologous recombination and Kan gene knockout; D represents the fluorescence characterization results of pDPV-ΔLORF2Rev transfected into DEF cells; and E represents the fluorescence characterization results of the purified DPV-ΔLORF2Rev strain. Figure 4 The figures show the PCR and RFLP identification results of DPV-ΔLORF2 and DPV-ΔLORF2-Rev strains in Example 1, where A represents the PCR identification result and B represents the RFLP identification result (a and b are respectively...). Eco R and Apa I is the simulation diagram, and c is the actual representation diagram). Figure 5The results are: Western blot identification of DPV-ΔLORF2 in Example 1 and in vitro growth kinetics of DPV-ΔLORF2 in Example 2, where A is the result of Western blot identification and B is the result of in vitro growth kinetics. Figure 6 The effect of LORF2 protein on poly(I:C)-activated IFN-β-Luc activity and IFN-β and ISG transcription levels is shown in Example 3. In this example, A represents IFN-β-Luc activity, and B represents IFN-β and ISG transcription levels. Figure 7 The effect of DPV-ΔLORF2 infection on the immune response of DEF or duck spleen in Example 3, where A is virus-infected DEF and B is virus-infected duck spleen; Figure 8 The figures show the body temperature, body weight, and survival rate of ducks infected with different doses of DPV-ΔLORF2 in Example 4. Column A represents 10... 3 TCID 50 Column B is 10 4 TCID 50 Column C is 10 5 TCID 50 ; Figure 9 The pathological changes observed during necropsy of the liver and spleen of ducks infected with DPV-ΔLORF2 in Example 4; Figure 10 The pathological changes observed during necropsy of the glandular gastroesophageal junction and cecum in ducks infected with DPV-ΔLORF2 in Example 4. Figure 11 The pathological changes observed during necropsy of the bursa of Fabricius and thymus in ducks infected with DPV-ΔLORF2 in Example 4; Figure 12 The image shows microscopic lesions in the duodenum and spleen of ducks infected with DPV-ΔLORF2 in Example 4. Figure 13 The body temperature, weight, and protection rate of DPV-ΔLORF2-immunized ducks after virulent virus challenge in Example 5; Figure 14 This is an anatomical lesion diagram of a DPV-ΔLORF2-immunized duck after 5 days of exposure to a virulent virus, as shown in Example 5. Figure 15 This is a microscopic pathological change image of a DPV-ΔLORF2-immunized duck 5 days after being challenged with a potent virus in Example 5. Figure 16 The viral load of DPV in various organs of ducks immunized with DPV-ΔLORF2 after challenge in Example 5; Figure 17 This refers to the detection of neutralizing antibodies against DPV-ΔLORF2 immunized ducks in Example 5. Detailed Implementation

[0021] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] 1. Experimental materials (1) Duck embryo, duckling and cell Nine-day-old duck embryos and 14-day-old ducklings tested negative for DPV and DPV antibodies; primary duck embryo fibroblasts (DEF) were prepared from nine-day-old duck embryos.

[0023] (2) Virus strains, strains, plasmids and vaccines The strain: Duck plague virus China strain (DPV-CHv, GenBank NO: JQ647509.1) was isolated, preserved, and provided by Cheng Anchun et al. from the College of Animal Science and Technology, Guizhou University. Its LD50 for 28-day-old ducks... 50 10 8 / 1mL.

[0024] Strain pDPV-CHv50 (GS1783): This strain was constructed, preserved, and provided by Cheng Anchun et al. from the College of Animal Science and Technology, Guizhou University, to obtain an infectious clone containing the complete genome sequence of DPV by uploading DPV-CHv to DEF cells for 50 generations, extracting viral genomic DNA, inserting it into a bacterial artificial chromosome plasmid, and then transforming it into GS1783 Escherichia coli competent cells.

[0025] Plasmids: pCAGGS, IFN-promoter-Luc (IFN-β-Luc) and pRL-TK were preserved and provided by Cheng Anchun et al., College of Animal Science and Technology, Guizhou University.

[0026] Vaccine: Duck plague attenuated live vaccine (Yabiying) (batch number: 202403) was purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.

[0027] (3) Antibody Rabbit anti-Flag / HA and mouse anti-HA monoclonal antibodies, mouse anti-Tubulin monoclonal antibody, and HRP-labeled goat anti-mouse / rabbit IgG were purchased from Proteintech; rabbit anti-LORF2 polyclonal antibody was prepared by this project.

[0028] (4) Primers The primers used in the examples are shown in the table below: Table 1 Primer Sequences

[0029] Example 1: Construction of duck plague virus LORF2 gene deletion and reversion strains Homologous recombination was performed in *E. coli* using the infectious DPV clone pDPV-CHv50 to replace the LORF2 gene with the Kan resistance gene, constructing pDPV-ΔLORF2-kan; then, by removing the Kan resistance gene, an infectious clone of the LORF2 gene deletion strain, pDPV-ΔLORF2, was constructed (see...). Figure 1 (See Figure A). Similarly, based on the infectious clone pDPV-ΔLORF2, homologous recombination was performed in *E. coli* to insert the Kan-LORF2 gene into the ΔLORF2 site, constructing pDPV-ΔLORF2-(LORF2-kan); then, by removing the Kan resistance gene, the missing LORF2 gene was restored, constructing the infectious clone pDPV-ΔLORF2Rev with restored LORF2 gene (see Figure A). Figure 1 (See Figure B in the middle). Finally, the LORF2 gene deletion strain DPV-ΔLORF2 and the restored strain DPV-ΔLORF2Rev were rescued from DEF.

[0030] The specific process includes the following steps: I. Construction of a duck plague virus LORF2 gene deletion strain 1. Amplification of the target fragment ΔLORF2-Kan Using pEP-Kan-S plasmid as a template, PCR amplification was performed using primers ΔLORF2-Kan-F / R as shown in SEQ ID NO. 1~2 to obtain a fragment containing homologous sequences at both ends of the LORF2 gene and the Kan resistance gene. The PCR amplification system consisted of 25 µL of 2× high-fidelity enzyme premix, 1 µL of template pEP-Kan-S plasmid, 1 µL each of ΔLORF2-Kan-F / R, and 22 µL of ddH2O. The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 30 cycles, and a final extension at 72℃ for 10 min. The amplified product was identified by agarose gel electrophoresis and the fragment was recovered to obtain the targeting fragment ΔLORF2-Kan.

[0031] 2. Homologous recombination knockout of the LORF2 gene (1) Preparation of pDPV-CHv50 (GS1783) electrocompetent states The frozen pDPV-CHv50 (GS1783) bacteria were cultured overnight at 30°C on LB / cm (LB containing chloramphenicol). A single colony was picked and inoculated into 5 mL of LB, and cultured overnight at 30°C with shaking to obtain the seed culture. 2.5 mL of the seed culture was added to 50 mL of LB / cm and cultured at 30°C with shaking until OD reached. 600 The value is around 0.5; after shaking in a 42℃ water bath for 15 min, immediately place on ice to cool for 20 min, centrifuge at 4℃ and 4500 r / min for 10 min to remove the supernatant; wash the bacterial pellet thoroughly with pre-cooled sterilized ddH2O, then centrifuge at 4℃ and 4500 r / min for 10 min, repeat centrifugation and washing 5 times to remove the supernatant; resuspend the bacterial pellet in 500 µL of pre-cooled ddH2O, aliquot into EP tubes, 100 µL per tube, to obtain pDPV-CHv50 (GS1783) electrocompetent cells, and store on ice for later use or at -80℃.

[0032] (2) Target shooting 200 ng of the targeting fragment ΔLORF2-Kan was added to the obtained pDPV-CHv50 (GS1783) electroporation competent cells, mixed thoroughly, and then transferred to a pre-cooled electroporation cuvette. Electroporation was performed at 2.5 kV, and the electroporation time was recorded. 800 µL of LB was added, and the mixture was thoroughly pipetted and transferred to an EP tube. The cells were incubated at 30°C with shaking for 1 h, then centrifuged at 4500 r / min for 2 min. The supernatant was discarded, and the cell pellet was resuspended in 80 µL of LB and evenly spread on LB / Cm+Kan (LB containing kanamycin and chloramphenicol) plates. The plates were incubated at 30°C for 48 h.

[0033] (3) Colony identification Using single colonies selected on LB / Cm+Kan plates as templates, colony identification was performed using LORF2-R (as shown in SEQ ID NO.4) and Kan-R (as shown in SEQ ID NO.6) (or LORF2-JD-F / R (as shown in SEQ ID NO.7~8)). The colony PCR identification system consisted of: 0.2 µL each of LORF2 downstream primer and Kan downstream primer, 0.2 µL of a single colony clone, 5 µL of 2×Mix, and ddH2O to 10 µL. The amplification program was: 98℃ for 5 min; 98℃ for 15 s, 55℃ for 15 s, 72℃ for 2 min, 30 cycles; 72℃ for 10 min. A positive clone pDPV-ΔLORF2-Kan, in which the Kan resistance gene replaced the LORF2 gene, was obtained.

[0034] 3. Homologous recombination to knock out the Kan resistance gene (1) Cultivation and screening Positive clone pDPV-ΔLORF2-Kan was inoculated into 2 mL LB / Cm+Kan and cultured overnight at 30°C with shaking to obtain seed culture. 10 µL of seed culture was added to 1 mL LB / Cm+Kan and cultured at 30°C until a slightly cloudy appearance was observed. The culture was centrifuged at 5000 r / min for 5 min, the supernatant was discarded, and 1 mL LB / Cm and 27 µL of 5 mol / L L-arabinose were added. The culture was then incubated at 42°C with shaking for 30 min, followed by incubation at 30°C with shaking for 2 h. 1 µL of the bacterial culture was thoroughly mixed with 200 µL LB and spread onto LB / Cm plates, then incubated at 30°C for 48 h.

[0035] (2) Colony identification Select the single colonies obtained in step (1) and perform PCR identification using LORF2-F / R as shown in SEQ ID NO.3~4. The PCR amplification system and amplification program are the same as in step 2 to obtain the infectious clone pDPV-ΔLORF2 of the duck plague virus LORF2 gene deletion strain.

[0036] 4. Virus rescue Infective clone DNA of pDPV-ΔLORF2 was extracted and transfected into DEF cells. Cell status was monitored daily. When obvious green fluorescent patches and obvious cytopathic effects were observed, samples were collected and new DEF cells were infected. Through intracellular homologous recombination, the DPV LORF2 gene deletion strain DPV-ΔLORF2 was finally obtained.

[0037] Results of constructing duck plague virus LORF2 gene-deleted strains: Colony PCR identification of the LORF2 gene knockout strain is shown in [link to relevant documentation]. Figure 2 Figure A (where M: DNA Marker; 1-8 are the target bacteria to be identified, of which 8 is a positive bacteria); PCR identification after pDPV-ΔLORF2-Kan homologous recombination to remove the Kan resistance gene is shown in Figure A. Figure 2 Figure B (where M: DNA Marker, 1-8 all have the Kan resistance gene removed, 9 is the target bacteria control); obvious green fluorescent spots were observed after pDPV-ΔLORF2 transfected into DEF cells. Figure 2 Figure C shows the final results of observing the DPV-ΔLORF2 strain with the DPV LORF2 gene deletion under a fluorescence microscope. Figure 2 Diagram D in the middle.

[0038] II. Construction of duck plague virus LORF2 gene revertant strain 1. Preparation of target fragments (1) Using pDPV-CHv50 as a template, the LORF2 homologous fragment was amplified using the LORF2-F / R primers shown in SEQ ID NO. 3~4. The PCR amplification system was as follows: 0.5 μL template, 5 μL PrimerStar Max DNA Polymerase, 0.3 μL each of LORF2-F / R, and ddH2O added to a final volume of 10 μL. The PCR amplification program was as follows: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 30 s, for 30 cycles; extension at 72℃ for 10 min.

[0039] (2) Using pEPKan-S as a template, the Kan homologous fragment was amplified using the Kan-F / R primers shown in SEQ ID NO. 5~6. The PCR amplification system was as follows: 0.5 μL template, 5 μL PrimerStar Max DNA Polymerase, 0.3 μL each of Kan-F / R primers, and ddH2O to a final volume of 10 μL. The PCR amplification program was as follows: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 30 s, for 30 cycles; 72℃ for 10 min.

[0040] (3) Using LORF2 homologous fragments and Kan homologous fragments as templates, and LORF2-F and Kan-R as primers, fusion PCR was performed to obtain the targeting fragment LORF2-Kan. The fusion PCR system was as follows: 5 μL PrimerStar Max DNA Polymerase, 0.5 μL each of LORF2-F and Kan-R, 0.5 μL each of LORF2 and Kan homologous fragments, and ddH2O to a final volume of 10 μL. The fusion PCR program was as follows: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 15 s, 72℃ for 2 min, for 30 cycles; 72℃ for 10 min.

[0041] 2. The first round of RED homologous recombination reverted to the LORF2 gene. (1) Preparation of pDPV-ΔLORF2 (GS1783) competent cells The pDPV-ΔLORF2 (GS1783) obtained in step one was prepared as a competent state using the same method as the pDPV-CHv50 (GS1783) electrotransfer competent state preparation method described above.

[0042] (2) Target shooting The specific method is the same as the targeting method used in the construction of the duck plague virus LORF2 gene deletion strain mentioned above.

[0043] (3) Colony identification Single colonies obtained in step (2) were selected and identified by colony PCR using the LORF2-F / R primers shown in SEQ ID NO. 3~4. Positive clones were named pDPV-ΔLORF2-Rev-Kan. The colony PCR identification system consisted of: 5 μL of 2×Mix, 0.2 μL each of primers LORF2-F / R, and an appropriate amount of colonies, with ddH2O added to a final volume of 10 μL. The colony PCR identification program was as follows: 94℃ for 3 min; 94℃ for 10 s, 55℃ for 15 s, 72℃ for 1 min, 30 cycles; 72℃ for 5 min.

[0044] 3. Second round of RED homologous recombination to remove the Kan resistance gene (1) Cultivation and screening The specific method is the same as the culture and screening method used in the construction of the duck plague virus LORF2 gene deletion strain mentioned above.

[0045] (2) Colony identification Colony PCR identification was performed on the colonies in (1) using the LORF2-JD-F / R primer pair shown in SEQ ID NO.7~8. The PCR identification system and procedure were the same as in step 2. The positive clones identified were infectious clones of the duck plague virus LORF2 gene revertant strain, pDPV-ΔLORF2Rev.

[0046] 4. Virus rescue The specific method is the same as the virus rescue in the construction of the duck plague virus LORF2 gene deletion strain mentioned above.

[0047] Results of constructing duck plague virus LORF2 gene revertant strain: Agarose gel electrophoresis results of PCR-amplified LORF2 homologous fragments, Kan homologous fragments, and their fusion fragments are shown in the figure. Figure 3 Figure A (where 1: Kan homologous fragment, 2: LORF2 homologous fragment, 3: fusion fragment); positive clones of the LORF2 gene target were identified by PCR. Figure 3 Figure B (where M: DNA Marker; 1-3 are the target bacteria to be identified, with 1 being a positive bacteria); Colony PCR identification of the infectious clone pDPV-ΔLORF2Rev of the duck plague virus LORF2 gene deletion revertant strain after homologous recombination knockout of the Kan gene is shown in Figure B). Figure 3 Figure C (where M: DNA Marker; 1-3 are Kan-positive bacteria with Kan removed); obvious green fluorescent spots were observed after pDPV-ΔLORF2Rev transfected into DEF cells. Figure 3 Figure D shows the final results of observing the DPV-ΔLORF2Rev strain with the DPV LORF2 gene deletion under a fluorescence microscope. Figure 3 Image E in Chinese.

[0048] III. Identification of the duck plague virus LORF2 gene deletion strain DPV-ΔLORF2 and the reverted strain DPV-ΔLORF2-Rev 1. Identification methods (1) PCR and sequencing identification The rescued DPV-ΔLORF2 and DPV-ΔLORF2-Rev were infected with DEF for 36 h, samples were collected, DNA was extracted as a template, and the target fragment was amplified using LORF2-JD-F / R as primers. The PCR products were sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0049] (2) RFLP identification use Eco R Ⅰ and Apa I. The pDPV-ΔLORF2, pDPV-ΔLORF2-Rev and pDPV-CHv50 plasmid DNAs were digested with enzymes respectively and incubated at 37°C for 1 h. The digestion products were subjected to agarose gel electrophoresis and gel imaging.

[0050] (3) Identification of proteins by Western blot 1 MOI DPV-ΔLORF2, DPV-ΔLORF2-Rev and DPV-CHv50 were used to infect DEF cells for 36 h. The supernatant was discarded, and the cells were washed three times with PBS. 100 μL of RIPA strong lysis buffer was added to each well, and the cells were lysed at 4°C for 30 min to obtain protein samples for Western blot analysis. SDS-PAGE was performed according to standard methods. PVDF membranes were transferred at 220 mA constant current for 2 h. The membranes were blocked overnight at 4°C with 5% skim milk powder. The membranes were incubated overnight at 4°C with rabbit anti-LORF2 polyclonal antibody (1:200) or rabbit anti-VP22 polyclonal antibody (1:500) as primary antibodies. The membranes were washed three times with TBST for 5 min each time. The membranes were incubated at room temperature for 1 h with HRP-labeled goat anti-rabbit IgG (1:5000) as secondary antibody. The membranes were washed three times with TBST for 5 min each time. ECL chemiluminescence was performed, and images were acquired using a universal Western blot imaging system.

[0051] 2. Appraisal Results (1) PCR and sequencing identification Viral DNA from DPV-ΔLORF2 and DPV-ΔLORF2-Rev was amplified by PCR using specific primers LORF2-JD-F / R, and the results are as follows: Figure 4As shown in Figure A (where M is the DNA Marker, 1 is the negative control, 2 is DPV-ΔLORF2-Rev, and 3 is DPV-ΔLORF2), the PCR product size of DPV-ΔLORF2 is 200 bp, and the PCR product size of DPV-ΔLORF2-Rev is 2093 bp, a difference of 1893 bp, which is consistent with the LORF2 sequence size; the sequencing results are also consistent with expectations, proving that the LORF2 gene was successfully deleted and restored in the obtained DPV-ΔLORF2 and DPV-ΔLORF2-Rev.

[0052] (2) RFLP identification The results are as follows Figure 4 As shown in Figure B, pDPV-CHv50, pDPV-ΔLORF2, and pDPV-ΔLORF2-Rev are subjected to... Eco After RⅠ restriction enzyme digestion, no differential bands were observed in any of the three plasmid groups. Figure 4 (c) in Figure B, and the expected ( Figure 4 This matches a) in Figure B; Apa After enzyme digestion, pDPV-ΔLORF2 specifically lost a band in the 4-5 kb region. Figure 4 (Green arrows in Figure B), while pDPV-CHv50 and pDPV-ΔLORF2-Rev maintain complete restriction enzyme digestion patterns, consistent with the simulated... Figure 1 To ( Figure 4 Figure B in the middle (b) indicates that pDPV-ΔLORF2 and pDPV-ΔLORF2-Rev were successfully constructed.

[0053] (3) Identification of proteins by Western blot The results are as follows Figure 5 As shown in Figure A, the LORF2 protein band could be detected in DEF infected by both DPV-ΔLORF2-Rev and DPV-CHv50, but not in the DPV-ΔLORF2 group. Furthermore, VP22 expression could be detected in all three viruses, indicating that the LORF2 protein was successfully deleted in DPV-ΔLORF2.

[0054] Example 2: In vitro growth kinetics of duck plague virus LORF2 gene deletion strain I. Experimental Methods DPV-ΔLORF2, DPV-ΔLORF2-Rev, and DPV-CHv50 were inoculated into 12-well DEF plates at a MOI of 0.01. After adsorption at 37°C for 2 h, the medium was replaced with maintenance medium containing 2% fetal bovine serum. Samples were collected at 12, 24, 48, 72, and 96 h post-infection and subjected to three freeze-thaw cycles. TCID was then serially diluted 10-fold. 50Viral titers were determined using the endpoint dilution method, and the number of cytopathic wells was counted and plotted as a line graph. Each experiment was performed in triplicate, and the data were analyzed for differences between groups using one-way ANOVA.

[0055] II. Experimental Results The results are as follows Figure 5 As shown in Figure B, the three viral strains, DPV-CHv50, DPV-ΔLORF2, and DPV-ΔLORF2-Rev, exhibited similar proliferation trends, all increasing from 12 h to 72 h and then decreasing after 96 h. However, the titer of DPV-ΔLORF2 was significantly lower than that of DPV-CHv50 and DPV-ΔLORF2-Rev, indicating that the LORF2 protein is beneficial for DPV proliferation.

[0056] Example 3: Transcriptional levels of IFN-β and ISGs in duck plague virus LORF2 gene deletion strain I. Experimental Methods (1) Effect of LORF2 protein on the activity of poly(I:C) activated IFN-β promoter Set up the following experimental groups: Positive control group: 200 ng poly(I:C), 200 ng IFN-β-Luc, 10 ng pRL-TK and 200 ng pCAGGS were co-transfected into DEF; Negative control group: 200 ng IFN-β-Luc, 10 ng pRL-TK and 400 ng pCAGGS were co-transfected into DEF; Experimental group: 200 ng poly(I:C), 200 ng IFN-β-Luc, 10 ng pRL-TK and 200 ng pCAGGS-LORF2-3Flag were co-transfected into DEF.

[0057] Samples were collected and analyzed 36 h post-transfection according to the instructions of the TransGold Dual-Luciferase Reporter Gene Assay Kit. The IFN-β-Luc value (A) and pRL-TK value (B) were measured. The relative IFN-β-Luc activity was obtained by dividing the A value by the B value and plotting the results using GraphPad. Each experiment was performed in triplicate, and the data were analyzed using one-way ANOVA to determine differences between groups.

[0058] (2) Effects of LORF2 protein on the transcriptional levels of poly(I:C) activated IFN-β and ISGs The group settings are the same as in step (1).

[0059] Thirty-six hours after transfection, total RNA samples were collected from cells using RNAiso Plus. Total RNA was extracted using the Trizol method, and genomic DNA was removed and reverse transcribed into cDNA according to the instructions of the Takara one-step reverse transcription kit. A quantitative PCR reaction was then performed. The qPCR reaction mixture consisted of: 5 μL TB Green® Premix Ex Taq™, 0.5 μL each of primers LORF2-DL-F / R (SEQ ID NO. 9~10), 1 μL cDNA template, and 3 μL ddH2O.

[0060] Finally, using 18S rRNA as an internal reference gene, the transcriptional levels of IFN-β, Mx, and OASL were detected. Each experiment was performed in triplicate, and the data were analyzed for differences between groups using one-way ANOVA.

[0061] (3) Effects of duck plague virus LORF2 gene deletion strain on IFN-β and ISG transcription levels DPV-ΔLORF2, DPV-ΔLORF2-Rev, and DPV-CHv50 with an MOI of 0.01 were infected with DEF for 48 h, and samples were collected. Simultaneously, 12 14-day-old ducklings were divided into four groups of three, and each group was intramuscularly injected with 10 mg / L of DPV-ΔLORF2. 2 TCID 50 Ducklings were euthanized after 72 hours with 1 mL / bird of each of DPV-ΔLORF2, DPV-ΔLORF2-Rev, DPV-CHv50 and DMEM. Spleen tissue was then collected.

[0062] Total RNA from DEF and spleen was extracted using standard methods, and reverse transcribed into cDNA according to step (2). The transcription levels of IFN-β, OASL, and Mx in each group were then detected by qPCR. Each experiment was performed in triplicate, and the data were analyzed using one-way ANOVA to determine inter-group differences.

[0063] II. Experimental Results (1) Effect of LORF2 protein on inhibiting the activity of poly(I:C) activated IFN-β promoter The results are as follows Figure 6 As shown in Figure A, compared with the pCAGGS transfection group, poly(I:C) treatment significantly enhanced the relative activity of IFN-β-Luc, increasing it by approximately 180-fold. However, when LORF2 protein was co-expressed, the stimulation of IFN-β-Luc activity by poly(I:C) was significantly weakened, with an inhibition of approximately 0.5-fold, indicating that LORF2 protein has the function of inhibiting the stimulation of IFN-β promoter activity by poly(I:C).

[0064] (2) Effects of LORF2 protein on the transcriptional levels of poly(I:C) activated IFN-β and ISGs The results are as follows Figure 6 As shown in Figure B, the LORF2 protein significantly inhibits the transcriptional levels of IFN-β, OASL, and Mx in the DEF, which are upregulated by poly(I:C). This indicates that the LORF2 protein has the ability to inhibit the production of host IFN-β and its downstream antiviral factors.

[0065] (3) Effects of duck plague virus LORF2 gene deletion strain on IFN-β and ISG transcription levels The results are as follows Figure 7 As shown, whether it is in vitro DEF ( Figure 7 (Figure A) or DEF in duck body ( Figure 7 (Figure B) The transcriptional levels of IFN-β, OASL, and Mx in the DPV-ΔLORF2 infection group were higher than those in the DPV-ΔLORF2-Rev and DPV-CHv50 infection groups, indicating that the LORF2 protein facilitates the function of DPV in inhibiting DEF and the production of immune factors in ducks.

[0066] Therefore, the duck plague virus LORF2 gene deletion strain can increase the level of immune factors in ducks.

[0067] Example 4: Evaluation of pathogenicity of duck plague virus LORF2 gene deletion strain in ducks I. Experimental Methods (1) Clinical symptoms of DPV-ΔLORF2 infection in ducks One hundred 14-day-old ducks were grouped and treated according to Table 2, and observed continuously for 10 days. The mortality rate, body temperature and weight changes of the ducks were recorded daily.

[0068] Table 2. Grouping and treatment of clinical symptoms in ducklings infected with DPV-ΔLORF2

[0069] (2) Pathological changes in necropsy of ducks infected with DPV-ΔLORF2 Thirty-six 14-day-old ducks were grouped and treated according to Table 3. Three ducks were euthanized on days 3, 5, and 9 after infection. Pathological changes in various tissues and organs were observed and photographed for record-keeping.

[0070] Table 3. Grouping and treatment of pathological changes observed in necropsy of ducks infected with DPV-ΔLORF2.

[0071] (3) Microscopic pathological changes in ducks infected with DPV-ΔLORF2 The spleen and duodenal tissues collected in step (2) were immediately fixed in 4% paraformaldehyde PBS fixative and stained with HE using conventional paraffin embedding techniques to prepare pathological sections for observation under an optical microscope.

[0072] II. Experimental Results (1) Clinical symptoms of infected ducks Experimental results are as follows Figure 8 As shown in the figure. The results showed that in ducklings treated with three different doses of DPV-CHv50 and DPV-ΔLORF2-Rev, the body temperature rose to above 43℃ on day 3 post-infection and persisted until day 5. On day 6, the body temperature of all surviving ducks returned to normal (40.5-42.5℃); 10 3 TCID 50 The body temperature of ducklings in the DPV-ΔLORF2 group remained within the normal range during the 10-day observation period, while... 4 and 10 5 TCID 50 Ducklings in the DPV-ΔLORF2 group also experienced elevated body temperature on day 3, but it did not rise above 43℃, and it was not until day 6 that their body temperature returned to normal. Ducks in the DMEM group maintained normal body temperature throughout the observation period. During the period of sustained high fever, ducks infected with different doses of DPV-CHv50 and DPV-ΔLORF2-Rev experienced decreased or even complete loss of appetite. On days 3 and 4 post-infection, their weight decreased instead of increasing, until day 6 when their body temperature returned to normal, after which their appetite gradually recovered and their weight steadily increased. 4 and 10 5 TCID 50 The DPV-ΔLORF2 group of ducklings did not gain weight as rapidly as the DMEM group, but no weight loss was observed; while 10 3 TCID 50 The weight gain rates of ducklings in the DPV-ΔLORF2 and DMEM groups were comparable. Mortality was recorded for each group during the observation period. Results showed no mortality in the DMEM and DPV-ΔLORF2 groups, while ducklings in the DPV-CHv50 and DPV-ΔLORF2-Rev groups died primarily between days 3 and 6. 3 10 4 10 5 TCID 50 The mortality rates of DPV-CHv50 were 20%, 40%, and 70%, respectively; 10 3 10 4 10 5 TCID 50 The mortality rates of DPV-ΔLORF2-Rev were 20%, 40%, and 60%, respectively. Figure 10The above results indicate that DPV-ΔLORF2 poses no lethal risk to ducks, but 10 3 TCID 50 Infection with the above doses can still cause ducks to exhibit clinical symptoms such as fever and loss of appetite.

[0073] (2) Pathological changes in infected ducks after necropsy The results are as follows Figures 9-11 As shown.

[0074] On day 3, no obvious ocular lesions were observed in any organ of the ducks in each group. At the peak of duck plague on day 5, significant hemorrhage was observed in all organs of both DPV-CHv50 and DPV-ΔLORF2-Rev, such as extensive hemorrhage in the liver, causing it to appear blackish-red. Figure 9 ); Splenic hemorrhage ( Figure 9 A bleeding band appeared at the junction of the dilated esophagus and the proventriculus. Figure 10 The duodenal wall is thinned and congested, and the cecum has unclear markings and numerous bleeding points. Figure 10 The bursa of Fabricius is covered with petechiae, and some parts show signs of atrophy. Figure 11 ); Thymic hemorrhage, severe atrophy ( Figure 11 No visible lesions were found in any organs in the DPV-ΔLORF2 and DMEM groups. After day 9, all ducks in all groups showed a significant reduction in visible lesions, but thymic atrophy and hemorrhage were still observed in the DPV-CHv50 and DPV-ΔLORF2-Rev groups. Figure 11 The results showed that DPV lacking LORF2 did not cause any ocular pathological changes in the organs of ducks.

[0075] (3) Histopathological changes in infected ducks The results are as follows Figure 12 As shown.

[0076] In ducks infected with DPV-ΔLORF2-Rev and DPV-CHv50, extensive necrosis of the epithelial cells at the top of the duodenal villi was observed, along with damage to the villus structure and partial villus loss, resulting in exposed lamina propria. Extensive necrosis of splenic lymphocytes was also observed, with the splenic sinuses filled with numerous hemocytes. In contrast, the spleen and duodenum of ducks infected with DPV-ΔLORF2 showed normal tissue structure, similar to the normal control group (DMEM), with no obvious microscopic lesions observed. These results indicate that DPV-ΔLORF2-Rev and DPV-CHv50 caused significant pathological damage to the spleen and duodenum of ducks, while DPV-ΔLORF2 did not cause significant damage.

[0077] Example 5: Evaluation of the immune effect of duck plague virus LORF2 gene deletion strain on ducks I. Experimental Methods 1. Protection of ducks immune to potent viral attacks by DPV-ΔLORF2 (1) Clinical symptoms Fifty 14-day-old ducks were randomly divided into 5 groups of 10 each, numbered 1-5. Groups 1-3 were injected intramuscularly with 1 mL of 10... 3 10 2 and 10 1 TCID 50 DPV-ΔLORF2, group 4 received 1 dose of commercial vaccine intramuscularly, and group 5 received 1 mL of DMEM intramuscularly. At 14 days, the LD50 was 100. 50 Ducks were challenged with the highly virulent DPV CHv and monitored for 10 days. Clinical symptoms such as body temperature, weight and protection rate were recorded.

[0078] (2) Pathological changes Twenty-seven 14-day-old ducks were randomly divided into three groups, numbered A through C, with nine ducks in each group. Group A received an intramuscular injection of 1 mL of immunized solution. 2 TCID 50 DPV-ΔLORF2, group B received one dose of commercial vaccine intramuscularly, and group C received 1 mL of DMEM intramuscularly. At 14 days, 100 LD... 50 Immunized ducks were challenged with a virulent DPV-CHv. Three ducks from each group were randomly euthanized on days 3, 5, and 9 after the challenge. Clinical and pathological changes in the heart, liver, spleen, thymus, bursa of Fabricius, cecum, duodenum, and rectum were photographed and recorded. Samples were collected and stored at -80°C for viral DNA detection. At the same time, the duodenum and spleen were collected, fixed, and prepared for pathological section observation.

[0079] 2. Detection of the clearance effect of DPV-ΔLORF2 immunized ducks against virulent DPV virus. DNA was extracted from the tissue samples collected and stored at -80℃ in step 1 (2). The LORF2 gene-based real-time PCR method was established using primers LORF2-DL-F / R (SEQ ID NO. 9~10) to analyze the effect of DPV virulence on immunized ducks in tissues and organs. Each group of experiments was performed three times biologically replicated. The data obtained were analyzed by two-way ANOVA to analyze the differences between groups and the clearance effect.

[0080] 3. Detection of antibody levels in ducks immunized with DPV-ΔLORF2 Nine 14-day-old ducks were randomly divided into three groups of three, numbered A through C. Each duck in group A received an intramuscular injection of 1 mL of 10 ml ... 2 TCID 50Group B received one dose of commercial duck plague vaccine (dPV-ΔLORF2 / mL) intramuscularly, while Group C received one mL of DMEM intramuscularly. Serum samples were collected at 1, 3, 5, 7, 14, and 21 days post-immunization to separate neutralizing antibody levels. Data were analyzed using one-way ANOVA to determine differences between groups.

[0081] II. Experimental Results 1. Protection of DPV-ΔLORF2-immunized ducks against DPV virulent attack. (1) Clinical symptoms of immunized ducks when challenged with a virulent virus Experimental results are as follows Figure 13 As shown. 10 2 and 10 3 TCID 50 The DPV-ΔLORF2 immunized group showed no duck mortality during the 10-day observation period after challenge, with a protection rate of 100%. 1 TCID 50 In the DPV-ΔLORF2 immunized group, one animal died on days 4 and 5 after infection with the virulent strain, resulting in a protection rate of 80%. No deaths occurred in the vaccine-treated group during the 10-day observation period, achieving a protection rate of 100%. In contrast, all animals in the non-immunized group died within 3-5 days after infection with the virulent strain. This indicates that... 2 TCID 50 Immunizing ducks with DPV-ΔLORF2 can completely protect them from attacks by a deadly and potent venom. And 10 2 and 10 3 TCID 50 Following DPV-ΔLORF2 challenge, the body temperature in the DPV-ΔLORF2 immunized group, similar to that in the commercial duck plague vaccine group, remained within the normal range (40.5-42.5℃). 1 TCID 50 In the DPV-ΔLORF2 immunized group, two ducks developed a body temperature of 43.8℃ after being challenged with a virulent strain; while in the DMEM group, the body temperature rose to >42.5℃ on day 2 after challenge, and remained around 43.5℃ on days 3 and 4. During the period of sustained high fever, the ducks experienced decreased appetite and weight loss instead of gain; whereas the commercial duck plague vaccine group, 10 2 and 10 3 TCID 50 The body weight of the DPV-ΔLORF2 immunized group continued to increase during the 10-day observation period. 1 TCID 50 The DPV-ΔLORF2 immunization group showed slower growth compared to the previous three groups during the 2-day period of fever elevation. This indicates that 10 2 TCID 50 DPV-ΔLORF2 immunization can protect ducks from developing any clinical symptoms when exposed to a potent venom.

[0082] (2) Pathological changes in immunized ducks when challenged with a virulent virus Pathological changes after dissection, such as Figure 14 As shown, in the non-immunized DMEM group, dead ducks exhibited a clear hemorrhagic band at the junction of the proventriculus and esophageal dilatation; the liver appeared congested; the spleen was dark red; the duodenum showed severe hemorrhage and thinning of the intestinal wall; the cecum was covered with hemorrhagic spots; and the thymus and bursa of Fabricius showed obvious hemorrhagic spots throughout. Meanwhile, 10... 2 TCID 50 No obvious pathological changes were observed in the DPV-ΔLORF2 immunization group and the commercial duck plague vaccine group.

[0083] Microscopic pathological changes such as Figure 15 As shown, in the non-immunized ducks, after challenge with a virulent strain, extensive necrosis of the epithelial cells at the top of the duodenal villi occurred, the villus structure was destroyed, and villi were lost in some areas, forming exposed lamina propria; a large number of lymphocytes died in the spleen, and the splenic sinuses were filled with a large number of hemocytes. In contrast, ducks immunized with DPV-ΔLORF2, like those immunized with the commercial duck plague vaccine, showed normal spleen and duodenal tissue structure after challenge with the virulent DPV strain, with no obvious microscopic lesions observed. These results indicate that DPV-ΔLORF2, like the commercial duck plague vaccine, can protect the tissues of immunized ducks from damage caused by the virulent strain.

[0084] 2. The effect of DPV-ΔLORF2-immunized ducks on clearing strong viruses. The results are as follows Figure 16 As shown, there was no difference in the virulent virus content between the immunized and non-immunized groups in the first 3 days after DPV virulent virus challenge. However, at 5 days, the DPV-ΔLORF2 immunized group, like the commercial duck plague vaccine group, showed significantly lower levels of virulent DPV virus in all tissues and organs except the liver compared to the non-immunized group. This indicates that 10 2 TCID 50 DPV-ΔLORF2 immunized ducks provide clearance of virulent DPV virus comparable to commercial vaccines.

[0085] 3. Neutralizing antibody levels in immunized ducks The results are as follows Figure 17 As shown, neutralizing antibodies could be detected 7 days post-immunization and continued to increase until 21 days. At 21 days post-immunization, the average neutralizing antibody titer in the DPV-ΔLORF2 immunization group reached 2... 3.86 The potency of commercially available duck plague vaccine can be 2. 3.8 The difference between the two was not significant. The results indicate that 10 2 TCID 50 DPV-ΔLORF2 immunization of ducks for 14 days resulted in neutralizing antibody levels comparable to those of a single dose of commercially available duck plague vaccine.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a duck plague virus LORF2 gene deletion strain, characterized in that, Includes the following steps: S1: Target fragment amplification Using pEP-Kan-S plasmid as a template, PCR amplification was performed using primers ΔLORF2-Kan-F / R as shown in SEQ ID NO.1~2 to obtain the ΔLORF2-Kan targeting fragment; S2: Homologous recombination to knock out the LORF2 gene The ΔLORF2-Kan targeting fragment was electroporated into pDPV-CHv50 competent cells. After antibiotic screening and PCR identification, the positive bacteria pDPV-ΔLORF2-Kan, in which the Kan resistance gene replaced the LORF2 gene, were obtained. S3: Homologous recombination to knock out the Kan resistance gene pDPV-ΔLORF2-Kan was induced and cultured, the Kan resistance gene was knocked out and PCR identification was performed to obtain the infectious clone pDPV-ΔLORF2 with the LORF2 gene missing; S4: Virus Rescue Infective clone pDPV-ΔLORF2 plasmid was extracted and transfected into DEF cells to obtain the LORF2 gene deletion strain DPV-ΔLORF2.

2. The method for constructing the duck plague virus LORF2 gene deletion strain according to claim 1, characterized in that, The PCR amplification system in S1 is a 50 µL system, which includes: 25 µL of 2× high-fidelity enzyme premix, 1 µL of template pEP-Kan-S plasmid, 1 µL each of ΔLORF2-Kan-F / R, and 22 µL of ddH2O.

3. The method for constructing the duck plague virus LORF2 gene deletion strain according to claim 1, characterized in that, The PCR amplification procedure in S1 is as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 30 cycles, and finally 72℃ extension for 10 min.

4. The method for constructing the duck plague virus LORF2 gene deletion strain according to claim 1, characterized in that, The primers used for PCR identification in S2 are LORF2-R as shown in SEQ ID NO.4 and Kan-R as shown in SEQ ID NO.6, or LORF2-JD-F / R as shown in SEQ ID NO.7~8.

5. The method for constructing a duck plague virus LORF2 gene deletion strain according to claim 1, characterized in that, The primers used for PCR identification in S3 are LORF2-F / R as shown in SEQ ID NO.3~4.

6. A duck plague virus LORF2 gene deletion strain, characterized in that, It is prepared by the construction method described in any one of claims 1 to 5.

7. The use of the duck plague virus LORF2 gene deletion strain according to claim 6 in the preparation of duck plague virus vaccine.

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