A carp herpesvirus type II orf150 gene deletion strain and a preparation method and application thereof

Abstract

The present application relates to a kind of carp herpesvirus II type ORF150 gene deletion strain and its preparation method and application, belong to aquatic animal disease prevention and control technical field.The present application carp herpesvirus II type ORF150 gene deletion strain preservation number is CCTCC NO:V202603, is preserved in China typical culture preservation center, and is named carp herpesvirus 2 type CyHV-2-SS24-Δ150-GFP Cyvirus cyprinidallo2 The virulence of the present application carp herpesvirus II type ORF150 gene deletion strain is significantly weakened, but still retains good immunogenicity, can induce the immune fish body to produce immune protection to carp herpesvirus II type.ORF Therefore, the present application carp herpesvirus II type ORF150 gene deletion strain can be used as live attenuated vaccine to carp herpesvirus II type, prevent koi and its variety in aquaculture from being infected by carp herpesvirus II type and lead to hematopoietic organ necrosis disease.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal disease prevention and control technology, and in particular to a carp herpesvirus type II ORF150 gene deletion strain, its preparation method, and its application. Background Technology

[0002] crucian carp ( Carassius spp. are important freshwater aquaculture fish, and the hybrid variety, heterozygous silver crucian carp ( C. auratus gibelio ) is a major aquaculture species in my country, and variant goldfish ( C. auratus auratus It is also an important ornamental fish species in the world. It is caused by carp herpesvirus type II (… Cyvirus cyprinidallo2 Herpesviral hematopoietic necrosis (HVHN) caused by CyHV-2 poses a significant obstacle to the green and sustainable development of the crucian carp and goldfish industry worldwide.

[0003] Carp herpesvirus type II belongs to the order Herpesvirales, family Alloherpesviridae, and genus Carp herpesvirus. Cyvirus It is an enveloped double-stranded DNA virus with a genome of about 290 kb enclosed in an icosahedral nucleocapsid. The size of a complete virus particle is usually 170 nm–200 nm. It can cause acute mass mortality in crucian carp and its variants. The main clinical symptoms include lethargy and anorexia, gill hemorrhage or paleness due to blood loss, abdominal swelling, and bleeding at the base of the fins and on the skin surface.

[0004] Vaccination strategies have proven to be highly effective and cost-effective in protecting fish from various viral infections, but currently there are no commercially available CyHV-2 vaccines for clinical application. Although many research teams both domestically and internationally have reported on the development of CyHV-2 vaccines, mainly in the form of inactivated vaccines, subunit vaccines, and DNA vaccines, most of these vaccines require the injection of high-concentration antigens one by one to achieve significant immune protection, while conventional attenuated vaccines carry the risk of virulence reversion.

[0005] Recombinant attenuated live vaccines are live vaccines with gene deletions or mutations. They reduce virulence by knocking out key virulence genes or related regulatory genes of the pathogen while retaining its immunogenicity. Therefore, recombinant attenuated live vaccines are not easily reversible in virulence under natural conditions, and due to their high stability, strong immunogenicity, and reliable safety, they are considered an important direction for the development of aquaculture vaccines. However, there are currently no research reports on CyHV-2 recombinant attenuated vaccines. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a carp herpesvirus type II ORF150 gene deletion strain, its preparation method, and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a carp herpesvirus type II ORF150 gene deletion strain, which has the accession number CCTCC NO:V202603 and is deposited at the China Center for Type Culture Collection, and is named carp herpesvirus type II CyHV-2-SS24-∆150-GFP.

[0008] This invention utilizes genetic engineering to delete the ORF150 gene in wild-type carp herpesvirus type II. Experiments have shown that, compared to the wild type, the virulence of this virus strain is significantly reduced, but it still retains good immunogenicity and can induce immune protection against carp herpesvirus type II in immunized fish. Therefore, the carp herpesvirus type II ORF150 gene-deleted strain of this invention can serve as a recombinant attenuated live vaccine against carp herpesvirus type II, preventing hematopoietic organ necrosis disease in aquaculture crucian carp and its variants.

[0009] The carp herpesvirus type II ORF150 gene deletion strain constructed in this invention is designated CyHV-2-SS24-∆150-GFP and was deposited at the China Center for Type Culture Collection (CCTCC) on March 26, 2026; deposit address: Wuhan University, Wuhan, China; postcode: 430072; accession number: CCTCC NO: V202603; name: carp herpesvirus type II CyHV-2-SS24-∆150-GFP; identification characteristics: Cyvirus cyprinidallo2 .

[0010] Secondly, the present invention provides a recombinant transfer plasmid, comprising a pUC-18 plasmid, upstream and downstream homologous arm elements of the carp herpesvirus type II ORF150 gene, and a foreign gene expression cassette element with a promoter; wherein the upstream and downstream homologous arm elements of the carp herpesvirus type II ORF150 gene, the foreign gene expression cassette element with a promoter, and the recombinant transfer plasmid are recombinant transfer plasmids. Bam HI and Eco Ligation of linear pUC18 plasmids digested with RI double enzyme.

[0011] This invention constructs a recombinant transfer plasmid for carp herpesvirus type II ORF150, which can induce homologous recombination of the wild-type carp herpesvirus type II genome in host cells, thereby causing the deletion or loss of function of the ORF150 gene on the wild-type carp herpesvirus type II genome, thus obtaining a carp herpesvirus type II ORF150 gene deletion strain.

[0012] Furthermore, the promoter is a CMV promoter; the exogenous gene is a selection marker gene.

[0013] Furthermore, the screening marker gene is a reporter gene or an antibiotic resistance gene.

[0014] Furthermore, the reporter gene is a green fluorescent protein gene or a luciferase gene; the antibiotic resistance gene is a neomycin resistance gene or a kanamycin resistance gene.

[0015] The present invention constructs a selection marker gene with a promoter in the viral vector to facilitate the screening and identification of recombinant viruses.

[0016] Furthermore, the screening marker gene used in the recombinant transfer plasmid constructed in this invention is the green fluorescent protein gene, and the recombinant transfer plasmid is designated as pUC18-∆150-GFP. The nucleotide sequence of the plasmid is shown in SEQ ID NO:12.

[0017] Thirdly, the present invention provides the use of the carp herpesvirus type II ORF150 gene deletion strain described in the first aspect or the recombinant transfer plasmid described in the second aspect in any of the following: (1) Application in the preparation of drugs for fish to resist carp herpesvirus type II infection; (2) Application in the preparation of drugs that relieve or treat any of the following in fish: lethargy and anorexia, gill hemorrhage, gill whitening due to blood loss, abdominal swelling, bleeding at the base of fins and on the skin surface, and necrosis of hematopoietic organs; (3) Application in the preparation of carp herpesvirus type II attenuated live vaccine; (4) Application in the preparation of reagents or kits for identifying infection or vaccination with wild-type carp herpesvirus type II; (5) Application in the preparation of viral vectors for expressing exogenous genes.

[0018] This invention involves injecting a carp herpesvirus type II ORF150 gene-deleted strain into hybrid silver carp, followed by challenge experiments with wild-type carp herpesvirus type II. The results demonstrate that the survival rate of the hybrid silver carp injected with the carp herpesvirus type II ORF150 gene-deleted strain was significantly higher than that of the negative control group. Therefore, the carp herpesvirus type II ORF150 gene-deleted strain of this invention possesses antiviral activity against wild-type carp herpesvirus type II and can be used as an antiviral drug against this virus infection. Furthermore, the carp herpesvirus type II ORF150 gene-deleted strain of this invention was constructed using the aforementioned recombinant transfer plasmid; therefore, this recombinant transfer plasmid can also be used for the preparation of drugs against carp herpesvirus type II infection in fish.

[0019] Since the clinical symptoms of fish (crucian carp) infected with carp herpesvirus type II include lethargy, anorexia, gill hemorrhage or paleness due to blood loss, abdominal swelling, hemorrhage at the base of the fins and on the skin surface, or necrosis of hematopoietic organs, the carp herpesvirus type II ORF150 gene deletion strain of this invention can resist carp herpesvirus type II infection. Therefore, the carp herpesvirus type II ORF150 gene deletion strain of this invention has the effect of alleviating or treating the above-mentioned clinical symptoms. Furthermore, the carp herpesvirus type II ORF150 gene deletion strain of this invention is constructed using the aforementioned recombinant transfer plasmid; therefore, this recombinant transfer plasmid can also be used for the preparation of drugs to alleviate or treat the above-mentioned clinical symptoms.

[0020] This invention involves injecting a carp herpesvirus type II ORF150 gene-deleted strain into hybrid silver carp, followed by a wild-type carp herpesvirus type II challenge experiment. The results showed that the survival rate (immunoprotection rate) of the hybrid silver carp injected with the carp herpesvirus type II ORF150 gene-deleted strain was 90% (experimental group 1) and 66.7% (experimental group 2), significantly higher than the negative control group. This indicates that the carp herpesvirus type II ORF150 gene-deleted strain possesses good immunogenicity. Simultaneously, safety experiments demonstrated that all crucian carp in both experimental groups injected with the virus strain of this invention survived, while the survival rate of crucian carp injected with wild-type virus was only 6.67%, indicating that the virulence of the virus strain of this invention has been significantly reduced, resulting in higher safety. Therefore, this invention can be used as a live attenuated vaccine for carp herpesvirus type II to prevent infection with carp herpesvirus type II. Furthermore, the carp herpesvirus type II ORF150 gene-deleted strain of this invention was constructed using the aforementioned recombinant transfer plasmid; therefore, this recombinant transfer plasmid can also be used for the preparation of a live attenuated carp herpesvirus type II vaccine.

[0021] Because the carp herpesvirus type II ORF150 gene deletion strain or recombinant transfer plasmid constructed in this invention contains expression cassette elements of a detection gene, the presence or absence of expression of this detection gene can be used to identify or distinguish between infection with wild-type carp herpesvirus type II and vaccination. For example, when the detection gene is a green fluorescent protein gene, the formation of green fluorescent spots in the host cells indicates vaccination; the absence of green fluorescent spots indicates infection with wild-type carp herpesvirus type II. Alternatively, the presence or absence of an EGFP nucleic acid sequence can be identified or distinguished using qPCR. Therefore, the carp herpesvirus type II ORF150 gene deletion strain or recombinant transfer plasmid constructed in this invention can be used to prepare reagents or kits for identifying infection with wild-type carp herpesvirus type II or vaccination.

[0022] The carp herpesvirus type II ORF150 gene-deleted strain of this invention knocks out the ORF150 gene, and its virulence has been significantly reduced after safety assessment. Furthermore, this deletion strain possesses typical viral vector characteristics: 1) It carries a foreign gene and can be packaged into viral particles: it can insert the target gene into its genome and form complete viral particles with infectivity in packaging cells; 2) It mediates the transfer and expression of foreign genes: it successfully introduces the target gene into target cells and achieves effective transcription and translation within the cells, exerting therapeutic or research functions; 3) It is non-lethal to animals: after modification, the viral virulence is significantly reduced, avoiding significant adverse reactions; 4) It has a suitable gene capacity: it can accommodate a certain length of foreign DNA fragment, with different vectors having different capacities. Therefore, the carp herpesvirus type II ORF150 gene-deleted strain of this invention can be used as a viral vector for expressing foreign genes. Since the carp herpesvirus type II ORF150 gene-deleted strain of this invention is constructed using the aforementioned recombinant transfer plasmid, this recombinant transfer plasmid can also be used as a vector for expressing foreign genes.

[0023] Fourthly, the present invention provides a drug for treating carp herpesvirus type II infection, comprising the carp herpesvirus type II ORF150 gene deletion strain described in the first aspect or the recombinant transfer plasmid described in the second aspect, and a pharmaceutically acceptable vector.

[0024] Fifthly, the present invention provides an immunogenic composition for preventing disease caused by carp herpesvirus type II, comprising the carp herpesvirus type II ORF150 gene deletion strain as described in the first aspect or the recombinant transfer plasmid as described in the second aspect; and a pharmaceutically or veterinarily acceptable vector, adjuvant or excipient.

[0025] Sixthly, the present invention provides a method for preparing the carp herpesvirus type II ORF150 gene deletion strain, comprising the following steps: (1) Amplify the upstream and downstream of the ORF150 gene of CyHV-2-SS24-WT strain to obtain upstream and downstream homologous arm elements; (2) The upstream and downstream homologous arm elements were ligated by overlapping extension PCR, double restriction sites were introduced, and seamless cloned homologous arm elements were ligated to obtain the modified upstream and downstream homologous arm elements of the ORF150 gene. (3) Using seamless cloning technology, the upstream and downstream homologous arm elements of the ORF150 gene obtained in step (2) are ligated to the linear pUC-18 plasmid vector to obtain the plasmid vector pUC18-UDF with upstream and downstream homologous arm elements of ORF150. (4) Using seamless cloning technology, the expression cassette element containing the promoter selection marker gene is ligated into the linear pUC18-UDF vector to obtain the carp herpesvirus type II ORF150 recombinant transfer plasmid described in the second aspect. (5) Transfect the recombinant transfer plasmid described in step (4) into the host cell, culture it, and then inoculate it with wild-type carp herpesvirus type II, so that the genome of wild-type carp herpesvirus type II and the recombinant transfer plasmid undergo homologous recombination in the host cell; after the cells are completely infected, collect the culture medium to obtain the viral fluid containing the carp herpesvirus type II ORF150 gene deletion strain, which is the carp herpesvirus type II ORF150 gene deletion strain.

[0026] This invention constructs a recombinant transfer plasmid containing homologous arms and selection markers, which achieves the replacement or deletion of target genes through homologous recombination within the host cell.

[0027] Furthermore, the amplification primers in step (1) include nucleotide sequences as shown in SEQ ID NO: 2-5.

[0028] Furthermore, the PCR primers in step (2) include nucleotide sequences as shown in SEQ ID NO: 6-9.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) High safety and good immunogenicity: This invention successfully constructed a carp herpesvirus type II ORF150 gene deletion strain (inactivated strain) for the first time. The virulence of this strain is significantly reduced, the safety is high, the genetic stability is not easy to relapse, and at the same time, it retains good immunogenicity and can effectively induce the host to produce comprehensive immune protection.

[0030] (2) The construction method is clear and reliable: This invention provides a specific construction method based on homologous recombination, with clear steps and strong operability. This method uses screening markers for initial screening and obtains genetically homozygous gene-deleted viruses through plaque purification, providing a stable technical path for the development of recombinant attenuated live vaccines.

[0031] (3) It provides a promising foundation for viral vectors: the recombinant transfer plasmid constructed from the genome of this deleted strain has been verified to integrate and express exogenous genes. This lays the foundation for its further development into an antigen expression vector.

[0032] (4) Prospects for industrial application: The deleted strain can be stably proliferated in suitable cell lines, which is convenient for large-scale preparation. Based on the immunogenicity of this strain, the dosage form can be one of the conventional dosage forms such as injection, oral or soaking agent suitable for aquaculture.

[0033] (5) Provides a molecular basis for differential diagnosis (DIVA): The deletion of the ORF150 gene provides a stable genetic marker for the attenuated strain. Based on this marker, a differential diagnostic method can be developed to distinguish between infection with wild-type carp herpesvirus type II and vaccination. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the plasmid map for the recombinant transfer plasmid pUC18-∆150-GFP.

[0035] Figure 2 The image shows the fluorescence microscopy results of different stages of CyHV-2-SS24-∆150-GFP infection of Ranchu goldfish swim bladder cells (RcGFSB).

[0036] Figure 3 A schematic diagram of the results of nested PCR identification of CyHV-2-SS24-∆150-GFP purity (where the lanes from left to right are: Lane 1: Marker; Lane 2: CyHV-2-SS24-∆150-GFP; Lane 3: CyHV-2-SS24-WT; Lane 4: Negative control RcGFSB).

[0037] Figure 4 This is a schematic diagram showing the survival rate of heterotrophic silver carp after challenged with CyHV-2-SS24-∆150-GFP or CyHV-2-SS24-WT, respectively.

[0038] Figure 5 A schematic diagram showing the survival rate of heterotrophic silver carp after immunization with CyHV-2-SS24-∆150-GFP for 21 days and challenged with CyHV-2-SS24-WT. Detailed Implementation

[0039] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0040] The carp herpesvirus type II ORF150 gene deletion strain constructed in this invention is designated CyHV-2-SS24-∆150-GFP and was deposited at the China Center for Type Culture Collection (CCTCC) on March 26, 2026. Deposit address: Wuhan University, Wuhan, China; Postcode: 430072; Deposit number: CCTCC NO: V202603; Name: Carp herpesvirus type II CyHV-2-SS24-∆150-GFP; Identification characteristics: Cyvirus cyprinidallo2 .

[0041] Example 1: Construction of the recombinant transfer plasmid of carp herpesvirus type II ORF150 1. Constructing upstream and downstream homologous arm elements of carp herpesvirus type II ORF150 The sequence of the ORF150 gene of CyHV-2-SS24-WT, a type II carp herpesvirus, is shown in the sequence listing SEQ ID NO:1: Using SnapGene software, upstream and downstream of CyHV-2-SS24-WT strain ORF150 were screened for strains that did not contain [the virus]. Bam HI and Eco The RI restriction site has a homologous arm of approximately 1 kb, and the following primers were designed for amplification: Upstream primer SS24-150UF-F used to amplify the upstream homologous arm of ORF150: 5′-GAATGCTATGGGGTTGGTGCCA-3′ (SEQ ID NO: 2) The downstream primer SS24-150UF-R used to amplify the upstream homologous arm of ORF150: 5′-CAATCCAGAGGCTCCGTCATT-3′ (SEQ ID NO:3) The upstream primer SS24-150DF-F used to amplify the downstream homologous arm of ORF150: 5′-CGAAGGCTTCCAACTTTGCCAC-3′ (SEQ ID NO:4) Downstream primer SS24-150DF-R used to amplify the downstream homologous arm of ORF150: 5′-CCACCGACAATGTCTGCGATCC-3′ (SEQ ID NO:5) Table 1 KOD Fx enzyme PCR amplification system Table 2 PCR amplification program After the PCR amplification products were separated and identified by agarose gel electrophoresis, they were purified by gel extraction to obtain the upstream and downstream homologous arm elements of carp herpesvirus type II ORF150, which were then stored at -20℃.

[0042] 2. Modification of upstream and downstream homologous arm elements of carp herpesvirus type II ORF150 The specific steps for linking upstream and downstream homologous arm elements of ORF150 and adding double restriction sites and seamless cloning homologous arms are as follows: Overlap extension PCR was used to ligate upstream and downstream homologous arm elements of ORF150, and insertion was performed between them. Bam HI and EcoThe RI double restriction site was used to add homologous arms at both ends for seamless cloning. The following primers were designed for amplification: Upstream primer SS24-150UF (used to add upstream seamless clonal homologous arms) Bam )-F: 5′-GCCTGCAGGTCGACTCTAGAGGATCGAATGCTATGGGGTTGGTGCCA-3′ (SEQ ID NO: 6) Used to add Bam The downstream primer SS24-150UF (+) for the HI restriction site Bam -R: 5′-GGAATTCGACAGCTCTGGATCCCAATCCAGAGGCTCCCGTCATT-3′ (SEQ ID NO:7) Used to add Eco The upstream primer for the RI restriction site is SS24-150DF (+). Eco -F: 5′-GGGATCCAGAGCTGTCGAATTCCGAAGGCTTCCAACTTTGCCAC-3′ (SEQ ID NO:8) Downstream primer SS24-150DF (used to add downstream seamless cloning homologous arms) Eco -R: 5′-ACAGCTATGACCATGATTACGAATTACCACCGACAATGTCTGCGATCC-3′ (SEQ ID NO: 9).

[0043] The modified upstream and downstream homologous arm elements of ORF150 were obtained using the amplification and purification methods described in step 1. The modified upstream and downstream homologous arm elements were then ligated by overlap extension PCR and amplified.

[0044] Table 3 Overlap Extension PCR System After the PCR amplification products were separated and identified by agarose gel electrophoresis, they were then purified by gel extraction to obtain the modified carp herpesvirus type II ORF150 upstream and downstream homologous arm elements (with the addition of double enzyme restriction sites and seamless upstream and downstream clonal homologous arms), which were stored at -20℃.

[0045] 3. Construct the carrier skeleton pUC18-UDF with upstream and downstream homologous arm elements of ORF150. Using seamless cloning technology, the upstream and downstream homologous arms of the modified carp herpesvirus type II ORF150 were ligated to... Bam HI and EcoAfter routine transformation, positive identification, and common plasmid extraction, the linear pUC-18 vector containing upstream and downstream homologous arm elements of ORF150 was obtained by digesting the linear pUC-18 vector with RI double enzymes and then stored at -20℃.

[0046] Table 4 Seamless cloning system and reaction conditions 4. Construct EGFP expression cassette elements with CMV promoters Using pEGFP-N3 plasmid as a template, the following primers were designed for PCR amplification, and seamless upstream and downstream cloning homologous arms were added: The upstream primer CMV-EGFP (used to add upstream seamless clonal homologous arms) Bam -F: 5′-TGACGGAGCCTCTGGATTGGGATCCGTTACATAACTTACGGTAAATGGCCC-3′ (SEQ ID NO: 10) The downstream primer CMV-EGFP (used to add a downstream seamless cloning homologous arm) is used. Bam -R: 5′-GCAAAGTTGGAAGCCTTCGGAATTTTACTTGTACAGTCGTCCATGCC-3′ (SEQ ID NO: 11) The modified (with added upstream and downstream seamless clonal homologous arms) EGFP expression cassette elements with CMV promoters were obtained using the amplification and purification methods in step 1 and stored at -20°C.

[0047] 5. Construction of the carp herpesvirus type II ORF150 recombinant transfer plasmid pUC18-∆150-GFP Using the seamless cloning technique from step 3, the EGFP expression cassette element with the CMV promoter from step 4 is ligated to the line that has undergone... Bam HI and Eco Following the RI double digestion step 3, the pUC18-UDF vector was transformed, positively identified, and endotoxin-free plasmid was extracted to obtain the endotoxin-free carp herpesvirus type II ORF150 recombinant transfer plasmid pUC18-∆150-GFP, which was then stored at -20℃. The recombinant transfer plasmid pUC18-∆150-GFP is shown in the image below. Figure 1 As shown, its nucleotide sequence is shown in the sequence listing SEQ ID NO:12.

[0048] Example 2: Construction and purification of carp herpesvirus type II ORF150 gene deletion strain 1. Constructing the CyHV-2-SS24-∆150-GFP strain with ORF150 deletion of carp herpesvirus type II. The recombinant transfer plasmid pUC18-∆150-GFP was transfected into RcGFSB cells of Ranchu goldfish swim bladders using an EZ3000Plus transfection reagent. One day in advance, an appropriate amount of cells were seeded into 6-well plates, and transient transfection was performed the next day when the cell confluence was approximately 70%. A transfection complex was prepared by adding 3 μg of recombinant transfer vector, 9 μL of transfection reagent, and 150 μL of Opti-MEM to each well. After standing at room temperature for 15 min, the complex was added to the cells (containing 2.5 mL of complete Opti-MEM culture medium), gently mixed, and incubated in a constant temperature incubator at 27°C and 5% CO2.

[0049] After culturing the cells for 6 h, the old culture medium was discarded, and 2.5 mL of fresh DMEM medium containing 2% FBS was added. Wild-type carp herpesvirus type II (CyHV-2-SS24-WT) (MOI=1) was then inoculated into the cells, allowing homologous recombination between the CyHV-2-SS24-WT genome and the recombinant transfer vector in RcGFSB cells. Once the cells were fully infected, the virus was harvested, subjected to two freeze-thaw cycles, and filtered through a 0.45 μm filter to obtain a viral solution containing the carp herpesvirus type II ORF150 gene deletion strain. This carp herpesvirus type II ORF150 gene deletion strain was designated CyHV-2-SS24-∆150-GFP.

[0050] The Ranchu goldfish swim bladder RcGFSB cells used in this embodiment were deposited at the Guangdong Provincial Microbial Culture Collection Center on March 25, 2026, with accession number GDMCC NO:68000, and classified as Ranchu goldfish cell line.

[0051] 2. Purification of CyHV-2-SS24-∆150-GFP The above-mentioned viral fluid was inoculated into new RcGFSB cells, such as... Figure 2 As shown, after CyHV-2-SS24-∆150-GFP formed relatively concentrated green fluorescent plaques on the cells, the culture medium was discarded, and the fluorescent plaques were carefully removed using a sterile toothpick and inoculated into new RcGFSB cells. This step was repeated multiple times to obtain purified CyHV-2-SS24-∆150-GFP.

[0052] 3. Determine the purity of CyHV-2-SS24-∆150-GFP After three blind passages of the purified CyHV-2-SS24-∆150-GFP in RcGFSB cells, the viral strain was collected and its DNA was extracted. Nested PCR identification was performed using primers designed on the outer and inner sides of the ORF150 gene: Outer-F (outer primer): 5′-AATGACGGAGCCTCTGGATT-3′ (SEQ ID NO: 13) The outer downstream primer SS24-150 Outer-R: 5′-CTACAATAGTGGCAAAGTTGGAAG-3′ (SEQ ID NO: 14) Inner upstream primer SS24-150 Nested-F: 5′-GTCCACAACAGCAGCAGAATCCA-3′ (SEQ ID NO: 15) Inner downstream primer SS24-150 Nested-R: 5′-GGCGTCCACATCATACTCCAGAAC-3′ (SEQ ID NO: 16) Identification was performed according to the PCR system and procedure in step 1 of Example 1: First, the outer primers were used for the first amplification, and then the amplicon amplified by the outer primers was used as a template for the second amplification by the inner primers. The products of the two amplifications were identified by agarose gel electrophoresis.

[0053] The results are as follows Figure 3 As shown, in the first amplification product, the template of the wild-type strain CyHV-2-SS24-WT amplified a wild-type positive band of 1808 bp, while the template of the purified recombinant strain CyHV-2-SS24-∆150-GFP only amplified a target band of 1395 bp, and the negative control showed no band. Furthermore, in the second amplification product, only the template of the wild-type strain CyHV-2-SS24-WT amplified a wild-type positive band of 275 bp, while other amplification products showed no band. This indicates that CyHV-2-SS24-∆150-GFP has been successfully purified.

[0054] Example 3: Safety assessment of carp herpesvirus type II ORF150 gene deletion strain 1. Experimental Methods: Healthy crucian carp (200±30 g) were selected for in vivo challenge experiments to evaluate the safety of CyHV-2-SS24-∆150-GFP as a recombinant attenuated live vaccine.

[0055] Two experimental groups (CyHV-2-SS24-∆150-GFP), one positive control group (CyHV-2-SS24-WT), and one negative control group (DMEM) were set up, with 30 fish in each group. Each group underwent the following treatments: Experimental group 1 (Undilution): 100 μL of CyHV-2-SS24-∆150-GFP virus stock solution (1011.49 copies / mL) was injected into the base of the pelvic fin. Experimental group 2 (10-fold dilution): 100 μL of CyHV-2-SS24-∆150-GFP virus dilution (10¹⁰.49 copies / mL) was injected into the base of the pelvic fin. Positive control group: 100 μL of CyHV-2-SS24-WT virus dilution (10¹⁰.78 copies / mL) was injected into the base of the pelvic fin. Negative control group (Mock-challenged): 100 μL of DMEM culture medium was injected into the base of the pelvic fin.

[0056] The above-mentioned treatment groups were kept in water at 25℃ for 21 days, and the survival rate was observed and recorded daily.

[0057] 2. Experimental Results: The results are as follows Figure 4 As shown, on day 21, all crucian carp in both experimental groups injected with CyHV-2-SS24-∆150-GFP survived, while the survival rate of crucian carp injected with CyHV-2-SS24-WT virus fluid was only 6.67%, indicating that the virulence of CyHV-2-SS24-∆150-GFP had been significantly reduced.

[0058] Example 4: Evaluation of the effectiveness of the carp herpesvirus type II ORF150 gene deletion strain 1. Experimental Methods: In vivo challenge experiments were conducted on the hybrid silver carp in experimental groups 1 and 2 (immunized with CyHV-2-SS24-∆150-GFP in Example 3) and the negative control group (immunized with a mock immunization, injecting 100 μL of DMEM culture medium into the base of the pelvic fin). Each fish was injected with 100 μL of CyHV-2-SS24-WT virus dilution (10¹⁰.86 copies / mL) into the base of its pelvic fin, serving as experimental group 1, experimental group 2, and the mock-immunized group, respectively, to evaluate the effectiveness of CyHV-2-SS24-∆150-GFP as a recombinant attenuated live vaccine. In addition, another 30 hybrid silver carp underwent the same challenge procedure as the experimental groups, serving as the mock-challenged control. All the fish were kept at 25℃ for 21 days, and the survival rate was observed and recorded daily.

[0059] 2. Experimental Results: The results are as follows Figure 5As shown, after being challenged with a high concentration of the wild-type strain (CyHV-2-SS24-WT), the immunoprotection rates of experimental groups 1 and 2 were 90% and 66.7%, respectively, while all crucian carp in the mock-immunized group died within 10 days, indicating that CyHV-2-SS24-∆150-GFP has good immunogenicity.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A carp herpesvirus type II ORF150 gene deletion strain, characterized in that, Its accession number is CCTCC NO:V202603, and it is deposited at the China Center for Type Culture Collection. It is named CyHV-2-SS24-∆150-GFP.

2. A recombinant transfer plasmid, characterized in that, The recombinant transfer plasmid includes the pUC-18 plasmid, upstream and downstream homologous arm elements of the carp herpesvirus type II ORF150 gene, and a foreign gene expression cassette element with a promoter; wherein the upstream and downstream homologous arm elements of the carp herpesvirus type II ORF150 gene, the foreign gene expression cassette element with a promoter, and the double-digested linear pUC18 plasmid are linked.

3. The recombinant transfer plasmid as described in claim 2, characterized in that, The promoter is the CMV promoter; the exogenous gene is a selection marker gene.

4. The recombinant transfer plasmid as described in claim 3, characterized in that, The screening marker gene is a reporter gene or an antibiotic resistance gene.

5. The use of the carp herpesvirus type II ORF150 gene deletion strain as described in claim 1 or the recombinant transfer plasmid as described in claim 2 in any of the following: (1) Application in the preparation of drugs for fish to prevent carp herpesvirus type II infection; (2) Application in the preparation of drugs that relieve or treat any of the following in fish: lethargy and anorexia, gill hemorrhage, gill whitening due to blood loss, abdominal swelling, bleeding at the base of fins and on the skin surface, and necrosis of hematopoietic organs; (3) Application in the preparation of carp herpesvirus type II attenuated live vaccine; (4) Application in the preparation of reagents or kits for identifying infection or vaccination with wild-type carp herpesvirus type II; (5) Application in the preparation of viral vectors for expressing exogenous genes.

6. A drug for treating carp herpesvirus type II infection, characterized in that, The drug comprises the carp herpesvirus type II ORF150 gene deletion strain of claim 1 or the recombinant transfer plasmid of claim 2, and a pharmaceutically acceptable vector.

7. An immunogenic composition for preventing disease caused by carp herpesvirus type II, characterized in that, The composition comprises the carp herpesvirus type II ORF150 gene deletion strain of claim 1 or the recombinant transfer plasmid of claim 2; and a pharmaceutically or veterinarily acceptable vector, adjuvant or excipient.

8. The method for preparing the carp herpesvirus type II ORF150 gene deletion strain as described in claim 1, characterized in that, The steps include the following: (1) Amplify the upstream and downstream of the ORF150 gene of CyHV-2-SS24-WT strain to obtain upstream and downstream homologous arm elements; (2) The upstream and downstream homologous arm elements were ligated by overlapping extension PCR, double restriction sites were introduced, and seamless cloned homologous arm elements were ligated to obtain the modified upstream and downstream homologous arm elements of the ORF150 gene. (3) Using seamless cloning technology, the upstream and downstream homologous arm elements of the ORF150 gene obtained in step (2) are ligated to the linear pUC-18 plasmid vector to obtain the plasmid vector pUC18-UDF with upstream and downstream homologous arm elements of ORF150. (4) Using seamless cloning technology, the selection marker gene expression cassette element containing the promoter is ligated to the linear pUC18-UDF vector of step (3) to obtain the carp herpesvirus type II ORF150 recombinant transfer plasmid as described in claim 2. (5) Transfect the recombinant transfer plasmid described in step (4) into the host cell, culture it, and then inoculate it with wild-type carp herpesvirus type II, so that the genome of wild-type carp herpesvirus type II and the recombinant transfer plasmid undergo homologous recombination in the host cell; after the cells are completely infected, collect the culture medium to obtain the viral fluid containing the carp herpesvirus type II ORF150 gene deletion strain, which is the carp herpesvirus type II ORF150 gene deletion strain.

9. The preparation method according to claim 8, characterized in that, The amplification primers in step (1) include nucleotide sequences as shown in SEQ ID NO: 2-5.

10. The preparation method according to claim 8, characterized in that, The PCR primers in step (2) include nucleotide sequences as shown in SEQ ID NO: 6-9.

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