Fish viral nervous necrosis recombinant adenovirus vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a recombinant adenovirus vaccine for viral nervous necrosis of fish as well as a preparation method and application of the recombinant adenovirus vaccine. The method for preparing the viral nervous necrosis disease recombinant adenovirus vaccine by utilizing the replication-defective recombinant adenovirus vector comprises the following specific steps: cloning nervous necrosis virus CP gene, constructing a Pshuttle-CMV-ECMV vector with a target gene, obtaining recombinant adenovirus plasmids, screening to obtain recombinant adenovirus and preparing the recombinant adenovirus vaccine. The prepared recombinant adenovirus vaccine for the viral nervous necrosis of the fish can be used for immunizing and preventing NNV virus infection in a juvenile fish stage in a soaking manner, the stability of the vaccine is good, and animal safety tests prove that the vaccine is safe, effective and convenient and simple in clinical use, and has a relatively good market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a recombinant adenovirus vaccine for fish viral neuronecrosis, and a preparation method and application thereof. Background Art

[0002] Fish Nervous Necrosis Virus (NNV) is a highly pathogenic RNA virus that causes significant economic losses to the global aquaculture industry. Viral nervous necrosis (VNN) caused by NNV virus infection is a Class II infectious disease that is prevalent in marine fish in almost all parts of the world except the Americas and Africa. The mortality rate for fry and young fish is extremely high, and in severe cases, the mortality rate can reach 100% within a week. In recent years, the number of infected fish species and the degree of damage have increased rapidly. NNV virus belongs to the genus Betanodavirus and the family Nodaviridae. It was first identified in Australia in the 1980s. The virus particles have a non-enveloped icosahedral morphology, are about 25 nanometers in size, and their genetic information is composed of two positive single-stranded RNAs, one is 2970 base pairs of RNA1, and the other is 1440 base pairs of RNA2, and these RNA molecules lack poly (A) tails at the 3' end. Although the NNV virus has been discovered for a long time, the strategy for preventing and controlling viral neuronecrosis in fish remains challenging. In theory, vertical transmission can be prevented by screening broodstock that do not carry the virus, but due to the sensitivity limitations of the detection technology, it is difficult to ensure that the screened broodstock are completely free of the virus. In addition, although disinfection with ozone and iodine reagents can reduce the horizontal transmission of the virus, the virus may exist on the surface of the fertilized eggs and inside the egg membrane at the same time, making it impossible to completely block the spread of the virus by surface disinfection alone.

[0003] Vaccination is a key means of preventing and controlling viral diseases. Although studies have shown that injection of recombinant viral capsid proteins can achieve certain immune protection in large-sized seedlings or adults, since NNV mainly infects the juvenile stage, the specific immune system of the fry is not fully developed at this time, making it difficult to effectively prevent and control it through traditional immunization methods. Therefore, there is currently no effective vaccine or prevention and control measures to control the occurrence of viral neuronecrosis in fish, resulting in the frequent occurrence of the disease in my country, which restricts the healthy development of the fish farming industry. Summary of the invention

[0004] The present invention provides a recombinant adenovirus vaccine for fish viral neuronecrosis and a preparation method and application thereof, in order to solve the problem of aquaculture losses caused by the susceptibility of fish to infection by NNV virus in the juvenile stage.

[0005] The present invention adopts the following technical scheme: a recombinant adenovirus vaccine for fish viral neuronecrosis, the antigen protein of the recombinant adenovirus vaccine contains the viral neuronecrosis virus CP gene, and the nucleotide sequence of the antigen of the recombinant adenovirus vaccine is shown in SEQ ID NO:1.

[0006] A method for preparing a recombinant adenovirus vaccine for fish viral neuronecrosis comprises the following steps: (1) Cloning of the neuronecrosis virus CP gene: The NNV-CP gene sequence of the virus was analyzed and the primers NNV-CP-F and NNV-CP-R were designed to amplify the cDNA of the NNV virus as a template. The homologous sequence and restriction site of the Pshuttle-CMV-ECMV vector were added to NNV-CP-F and NNV-CP-R respectively; (2) Construction of Pshuttle-CMV-ECMV vector carrying NNV-CP: The cloned NNV-CP gene was cloned into the Pshuttle-CMV-ECMV vector by homologous recombination technology to construct a Pshuttle-CMV-ECMV vector carrying the NNV-CP gene, named Pshuttle-CMV-ECMV / NNV-CP; (3) Obtaining recombinant adenovirus plasmid: homologously recombining the obtained recombinant adenovirus vector Pshuttle-CMV-ECMV / NNV-CP with the PAd-easy-1 vector in Escherichia coli BJ5183 to construct a recombinant adenovirus plasmid; (4) Screening and obtaining recombinant adenovirus: The screened recombinant adenovirus positive plasmid was digested with Pac Ⅰ and used to transfect HEK293 cells to obtain recombinant adenovirus carrying the NNV-CP gene; (5) Preparation of recombinant adenovirus vaccine for viral neuropathy: After the harvested recombinant adenovirus carrying the NNV-CP gene is subcultured, the expression of the target antigen protein is detected. The recombinant adenovirus that can express the protein after detection is inoculated into HEK293 cells for multiple subcultures, repeated freezing and thawing, and the virus titer is measured. The high-titer recombinant adenovirus is used to prepare the recombinant adenovirus vaccine for viral neuropathy.

[0007] Furthermore, the nucleotide sequence of the primer NNV-CP-F is shown in SEQ ID NO:2.

[0008] Furthermore, the nucleotide sequence of the primer NNV-CP-R is shown in SEQ ID NO:3.

[0009] Furthermore, the high titer of the recombinant adenovirus should be greater than 1×10 8 mL -1 TCID50.

[0010] Furthermore, 1 ml of the high-titer recombinant adenovirus in step (5) is used to infect cultured HEK293 cells. After 72 hours of infection, the HEK293 cells and the supernatant are repeatedly frozen and thawed three times, and then centrifuged at 3000-4000 rpm / min for 8-10 minutes to obtain the fish rhabdovirus recombinant adenovirus vaccine.

[0011] The invention discloses an application of a recombinant adenovirus vaccine for fish viral neuronecrosis, which is used for preparing a therapeutic drug for viral neuronecrosis caused by fish neuronecrosis virus.

[0012] The invention discloses an application of a recombinant adenovirus vaccine for fish viral neuronecrosis, which is used for preparing an immersion preparation for resisting fish neuronecrosis virus.

[0013] The CP gene of NV virus plays a key role in the infection process of the virus. CP is the only structural protein of NNV virus particles. It not only participates in the assembly of virus particles, but also contains multiple antigenic epitopes that can induce immune responses in the host. In addition, NNV-CP protein can antagonize the host's immune response through various mechanisms, such as degrading key immune proteins of the host through the ubiquitin-proteasome pathway, thereby inhibiting the production of type I interferon. Since the NNV-CP gene plays an important role in the life cycle of the virus and the host's immune response, it has become an important target for vaccine development. By expressing the CP gene of NNV in vitro, recombinant proteins with good immunogenicity can be prepared, which is a key gene for the development of effective vaccines. For example, studies have successfully obtained recombinant CP protein by expressing the CP gene of NNV in Escherichia coli, and confirmed that it can induce protective immune responses. However, the antigenicity of the NNV-CP gene may differ in vivo and in vitro, which indicates that vaccine design needs to consider the antigenic characteristics of the virus in the host. In addition, the stability of the NNV-CP gene and the antigenic epitopes on the surface of the virus particles are crucial to the effectiveness of the vaccine. Existing studies have shown that the antigenic epitopes on the surface of NNV virus particles may change in heat treatment or specific buffers, thereby affecting the antigenicity and infectivity of the virus. In summary, the NNV-CP gene plays a key role in the infection mechanism of the virus, host immune escape, and vaccine development. The present invention constructs a recombinant adenovirus vaccine by cloning the NNV-CP gene, which is of great significance for the preparation of fish antiviral drugs and disease prevention and control.

[0014] The present invention has the following beneficial effects: (1) The present invention amplifies the viral neuronecrosis virus CP gene, connects it to the Pshuttle-CMV-ECMV vector, and after PCR screening is positive, homologous recombination is carried out with the PAd-easy-1 backbone vector to construct a replication-defective recombinant adenovirus plasmid, and the cells are transfected with the plasmid to obtain a replication-defective recombinant adenovirus containing the target gene; the recombinant adenovirus after detection is inoculated into HEK293 cells for multiple passages, and a high-titer recombinant adenovirus is selected to prepare a recombinant adenovirus vaccine for viral neuronecrosis, and the prepared vaccine has high stability.

[0015] (2) The recombinant adenovirus vaccine for fish viral neuronecrosis prepared by the present invention can be immunized by immersion, and can be used for immunization in the fish seedling stage. The immune protection rate is as high as 70% or more, and the immune effect is good. It overcomes the problem that the traditional injection vaccine is limited in use in the juvenile stage, has good application prospects, and can bring better economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The construction of the NNV-CP gene recombinant adenovirus vector of the present invention is as follows: A: Identification of NNV-CP gene PCR products: Lane 1, DNA Maker; Lane 2: NNV-CP gene PCR amplification product; Lane 3: blank control; B: PCR amplification and double restriction digestion results of the recombinant plasmid: Lane 1, DNA Maker; Lane 2, Sal I and XhoⅠ double restriction digestion results; Lane 3, PCR amplification results; Lane 4, empty vector PCR amplification results.

[0017] C: Identification of recombinant adenovirus vector: Lane 1, DNA Maker; Lane 2, Pac I digestion result of recombinant adenovirus plasmid; Lane 3, PCR amplification result of CP gene; Lane 4, blank control.

[0018] Figure 2 The green fluorescence expression results of the NNV-CP recombinant adenovirus vaccine of the present invention are shown in Figure 1: A: GFP expression in HEK293 cells infected with the recombinant adenovirus; B: HEK293 cell blank control group.

[0019] Figure 3 The Western-blot detection results of the NNV-CP protein expression of the NNV-CP recombinant adenovirus of the present invention are as follows: Lane 1, the expression results of the recombinant adenovirus CP protein harvested from HEK293 cells; Lane 2, the recombinant empty vector virus control group harvested from HEK293 cells; Lane 3, the HEK293 cell blank control group.

[0020] Figure 4It is the survival rate of grouper after being treated with the NNV-CP recombinant adenovirus vaccine of the present invention for 7 days.

[0021] Figure 5 This is a diagram showing the effect of the NNV-CP recombinant adenovirus vaccine of the present invention in preventing and treating viral neuronecrosis in grouper. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0023] Human embryonic kidney 293 cells (HEK293 cells) adhered to the cell culture medium and were cultured in a constant temperature incubator at 5% CO2 and 37°C. DMEM medium (Gibco) containing 10% fetal bovine serum, 100U / ml penicillin and 100mg / ml streptomycin was used as the cell culture medium for 293 cells; DMEM medium containing only 2% fetal bovine serum was used as the maintenance medium for HEK293 cells during virus culture.

[0024] The Nervous Necrosis Virus (NNV) in the present invention is stored in the National Aquatic Animal Pathogen Bank.

[0025] Example 1: Construction of NNV-CP gene recombinant adenovirus vector: (1) Cloning of the CP gene of neuronecrosis virus: First, the DNA template of NNV-CP was amplified by PCR using primers NNV-CP-F and NNV-CP-R with NNV virus cDNA as template. The sequence of primer NNV-CP-F is shown in SEQ ID NO: 2: 5'-ATTTGCGGCCGCGCCACCATGGTACGCAAAGGTGAGAAG -3' The sequence of primer NNV-CP-R is shown in SEQ ID NO: 3: 5'-CCGCTCGAGGTTTTCCGAGTCAACCCTG-3', The specific steps are as follows: NNV-CP-F 1μL (10μM), NNV-CP-R 1μL (10μM), NNV virus cDNA 1μL, high-fidelity enzyme premix (Novozyme) 10μL, add nuclease-free pure water to 20μL, and perform PCR amplification according to the following program, 1 cycle (95℃ 3min), 35 cycles (95℃ 10s, 58℃ 30s, 72℃ 30s), 1 cycle (72℃ 5min), and store at 4℃. After the PCR amplification, agarose electrophoresis was performed to verify the results of NNV-CP gene PCR. After obtaining a single NNV-CP gene target fragment, the above PCR product was purified using a DNA purification recovery kit (Tiangen); the results are as follows. Figure 1 As shown in A.

[0026] like Figure 1 The results showed that the NNV-CP gene DNA template was successfully amplified.

[0027] (2) Construction of Pshuttle-CMV-ECMV vector with target gene (NNV-CP): The purified NNV-CP gene was double-digested with Not I and Xho I restriction endonucleases to produce compatible sticky ends. The Pshuttle-CMV-EGFP vector was also double-digested with Not I and Xho I restriction endonucleases to match the ends of the NNV-CP gene. The digested NNV-CP gene and the Pshuttle-CMV-EGFP vector were ligated overnight at 16°C using T4 DNA ligase to form a recombinant DNA molecule. The ligation product was transformed into Escherichia coli DH5α competent cells. The transformed cells were spread on LB agar plates containing kanamycin and inverted for 12 to 16 hours. Smaller single colonies were selected from the cultured plates and transferred to liquid culture medium containing kanamycin for expansion culture for 12 hours. PCR identification was performed on the expanded culture culture to confirm the insertion of the NNV-CP target gene. The bacterial liquid with the same PCR identification results as the target size was further expanded and cultured, and then the plasmid DNA was extracted using a plasmid extraction kit (Tian Gen) and sequenced to verify the sequence correctness of the recombinant plasmid; the results are as follows Figure 1 As shown in B.

[0028] Depend on Figure 1 As shown in B, the NNV-CP fragment was successfully homologously recombined into the Pshuttle-CMV-EGFP vector.

[0029] (3) Obtaining the Pshuttle-CMV-EGFP / NNV-CP recombinant adenovirus plasmid: First, the Pshuttle-CMV-EGFP / NNV-CP vector was digested with Pme I to obtain a linearized vector. Subsequently, the linearized Pshuttle-CMV-EGFP / NNV-CP vector was transformed into BJ5183 competent cells, which already contained the PAd-easy-1 auxiliary vector, by heat shock. The transformation operation included mixing 5 μL of purified linearized vector with 50 μL of BJ5183 competent cells, followed by heat shock treatment at 42°C for 80 seconds. After heat shock, the recombinant mixture was spread on an LB plate containing 100 μg / mL kanamycin for screening. The PCR product was detected by 0.8% agarose gel electrophoresis to confirm the insertion of the target gene. After a large number of positive products were cultured, the plasmid DNA was extracted and digested with Pac I restriction endonuclease to verify the correctness of the recombinant adenovirus plasmid. The selected positive plasmids were further transformed into Stbl3 competent cells by heat shock method to obtain a large amount of high-quality plasmid DNA. The selected positive clones were again digested with Pac I to ensure that the plasmid construction was correct. Finally, the positive digestion products were recovered and prepared for the next transfection experiment; the results are shown in Figure 1 As shown in C.

[0030] like Figure 1 As shown in the results of C, the Pshuttle-CMV-EGFP / NNV-CP recombinant adenovirus plasmid was successfully obtained.

[0031] (4) Screening to obtain NNV-CP recombinant adenovirus: The recombinant adenovirus positive plasmid obtained in the previous step was digested with Pac I to remove the Ori original and kanamycin resistance gene, thereby exposing the adenovirus inverted terminal repeat sequence (ITR) and packaging signal. Then, the linearized recombinant adenovirus plasmid was transfected into HEK293 cells using liposomes.

[0032] The specific steps are as follows: 24 hours before transfection, HEK293 cells in the logarithmic growth phase were taken, the cells were digested with trypsin digestion solution, and then diluted with cell culture medium to a cell density of 1×10 7 cells / ml, form a cell suspension, add the above cell suspension at 5ml / bottle into 25cm 2The cells were placed in a cell culture flask and cultured in a cell culture incubator with a CO2 volume content of 5% and a temperature of 37°C. When the cell adhesion rate reached 60~70%, transfection was performed. 30μL LipofectamineTM 3000 was mixed with 900μL DMEM cell culture medium, and 4μg of linearized recombinant adenovirus plasmid was added. After incubation at room temperature for 20 minutes, the mixture was added to the cells and cultured at 37°C for 4 hours. Subsequently, the culture medium was removed and supplemented with 10mL of cell maintenance medium containing 10% FBS, and the culture was continued. The transfection efficiency was evaluated by monitoring intracellular GFP expression and cytopathic effect (CPE). When a large amount of GFP expression was observed in the cells, 5mL of the upper cell maintenance medium was gently removed, and the remaining culture medium was collected for virus passaging and amplification. The results are as follows. Figure 2 shown.

[0033] Depend on Figure 2 The results showed that a large amount of GFP fluorescence could be observed in HEK293 cells transfected with adenovirus vaccine plasmid NNV-CP 48 hours after transfection, while no GFP fluorescence was observed in negative control HEK293 cells, indicating that the adenovirus vaccine plasmid NNV-CP was successfully constructed.

[0034] (5) Preparation of recombinant adenovirus vaccine for viral neuropathy: HEK293 cells in the logarithmic growth phase were digested with trypsin and then diluted with cell culture medium to a cell density of 1×10 7 cells / ml, form a cell suspension, add the above cell suspension at 5ml / bottle into 25cm 2The cells were placed in a cell culture flask and cultured in a cell culture incubator with a CO2 volume content of 5% and a temperature of 37°C. When the cell adhesion rate reached 60-70%, the culture medium was removed and the culture medium containing the virus collected in the previous step was added. When a large amount of GFP expression was observed in the cells, the virus solution of HEK293 cells was collected and the cells were then lysed using RIPA lysis buffer. After lysis, the protein sample was broken by ultrasound and centrifuged at 12000 rpm for 10 minutes at 4°C to separate the supernatant. The supernatant was mixed with 6× Loading Buffer and then heated at 95°C for 10 minutes to denature the protein. Next, the treated protein sample was loaded into the SDS-PAGE electrophoresis gel well for electrophoresis separation. The electrophoresis conditions were set to 80V for 1 hour, and then the voltage was increased to 120V until the end of electrophoresis. Using β-actin as an internal reference, the protein bands to be tested in the gel were cut according to the protein molecular weight standard, and these protein bands were transferred to the PVDF membrane under the transfer condition of 200mA current for 2 hours. After the membrane transfer was completed, the PVDF membrane was blocked in a 5% skim milk powder solution for 1 hour to reduce nonspecific binding. After blocking, the membrane was incubated with a 1:1000 diluted GFP antibody overnight to specifically bind to the target protein. The next day, the membrane was washed 3 times with PBST buffer for 10 minutes each time to remove unbound antibodies. After that, the membrane was incubated with a 1:2000 diluted goat anti-mouse secondary antibody for 2 hours, and then washed 3 times with PBST buffer for 10 minutes each time to remove excess secondary antibody. Finally, the developer was added and the chemiluminescent reaction on the membrane was detected to evaluate the expression of the target protein in the recombinant adenovirus.

[0035] For recombinant adenovirus that can stably express the target protein, it was inoculated into HEK293 cells and passaged multiple times. After each harvest of the virus solution, it was repeatedly frozen and thawed 2 to 3 times to release the virus particles. By measurement, when the titer of the recombinant virus reached 1×10 8 mL -1 When TCID50 was 0.04, the recombinant adenovirus stock solution was used to prepare a recombinant adenovirus vaccine for viral neuropathy. HEK293 cells in the logarithmic growth phase were taken and digested with trypsin digestion solution, and then diluted with cell culture medium to a cell density of 1×10 7 cells / ml, form a cell suspension, add the above cell suspension at 5ml / bottle into 25cm 2The cell culture flask was placed in a cell culture incubator for culture. The CO2 volume content in the cell culture incubator was 5% and the temperature was 37°C. When the cell adhesion rate reached 60-70%, the culture medium was removed, and 1 ml of the recombinant adenovirus collected in the previous step was added to infect the cultured HEK293 cells. After culturing for 1 hour, 4 ml of HEK293 cell maintenance medium was added. After culturing for 72 hours, the above HEK293 cells and the supernatant were repeatedly frozen and thawed 3 times, and then centrifuged at 4000 rpm / min for 10 minutes. The supernatant was taken. The supernatant was the recombinant adenovirus vaccine for viral neuronecrosis. The recombinant adenovirus vaccine for viral neuronecrosis includes a recombinant adenovirus capable of expressing the NNV virus CP gene and HEK293 cell maintenance medium. The results are as follows Figure 3 shown.

[0036] Depend on Figure 3 The results showed that HEK293 cells infected with NNV-CP recombinant adenovirus vaccine could stably express NNV-CP protein, while HEK293 cells infected with recombinant empty vector adenovirus and blank HEK293 cells did not express NNV-CP protein. This indicates that the NNV-CP recombinant adenovirus vaccine can successfully express the target protein NNV-CP, and the NNV-CP recombinant adenovirus vaccine was successfully constructed.

[0037] Example 2: Safety and efficacy verification of recombinant adenovirus vaccine for viral neuronecrosis: In order to comprehensively evaluate the safety of the recombinant adenovirus vaccine in the present invention for preventing viral neuronecrosis and ensure the safety of its clinical application, a series of safety tests were conducted on the vaccine in accordance with the provisions of the Chinese Veterinary Pharmacopoeia. Groupers weighing 10 g were used as experimental animals, and the titer of the recombinant adenovirus vaccine used was 1×10 9 mL -1 TCID50.

[0038] In the experimental design, the first group of groupers were immersed in artificial seawater diluted with 1×10 9 mL -1 The TCID50 recombinant adenovirus vaccine was used to prepare a recombinant adenovirus vaccine soaking preparation capable of immersion immunization, and the recombinant adenovirus vaccine soaking preparation was used for immunization for 15 minutes; the second group of groupers were inoculated with 0.1 mL of the recombinant adenovirus vaccine by injection; the third group of groupers served as an unvaccinated control group. During the experiment, all experimental groups of groupers were closely monitored for clinical adverse reactions, and the number of dead fish was recorded every day within 7 days after immunization. In addition, the length and weight changes of each group of fish were regularly measured and recorded within 60 days after vaccination. The results are as follows Figure 4 It is knowable.

[0039] according to Figure 4 The results shown in the figure show that, while the grouper in the control group was in good growth condition, no fish deaths were observed in the first group, which was vaccinated by immersion, and no clinical adverse reactions were found in the second group, which was vaccinated by injection, within 7 days. Moreover, compared with the control group, there was no significant difference in the length and weight of the fish in the two vaccination groups after 60 days. These results show that the recombinant adenovirus vaccine has a high safety, and the immunization effect of immersion immunization is equivalent to that of injection vaccination. The experimental results also provide a scientific basis for its clinical use.

[0040] Example 3: Application of recombinant adenovirus vaccine for viral neuronecrosis: Groupers weighing about 10 grams were selected and vaccinated by immersion in artificial seawater at a dilution ratio of 1×10 9 mL -1 TCID50 recombinant adenovirus vaccine was prepared to obtain a recombinant adenovirus vaccine soaking preparation capable of soaking immunization. The recombinant adenovirus vaccine soaking preparation was used for immunization for 15 minutes. In order to ensure the control of the experiment, a negative control group without soaking vaccine was also set up. The experimental fish were cultured in a circulating water aquaculture system at 28°C. After a 28-day immunization period, all groupers were intraperitoneally injected with 0.1 ml of 1×10 5 TCID50 / mL of NNV virus. During the subsequent observation period, the survival and death of groupers in each group were recorded every day, and survival curves were drawn based on these data to evaluate the protective effect of the vaccine. Figure 5 shown.

[0041] according to Figure 5 The experimental results shown in the figure show that the mortality rate of grouper vaccinated with the NNV-CP recombinant adenovirus vaccine was about 30% during the 20-day observation period. In contrast, the mortality rate of grouper in the negative control group that was not vaccinated reached 100% during the same period of time. This significant difference indicates that the NNV-CP recombinant adenovirus vaccine vaccinated by immersion has a 70% protective efficacy against NNV virus infection in grouper.

[0042] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A recombinant adenovirus vaccine for fish viral neuronecrosis, characterized in that: The antigen protein of the recombinant adenovirus vaccine contains the viral neuronecrosis virus CP gene, and the nucleotide sequence of the antigen of the recombinant adenovirus vaccine is shown in SEQ ID NO:

1.

2. The method for preparing the recombinant adenovirus vaccine for fish viral neuronecrosis according to claim 1, characterized in that: The steps include: (1) Cloning of the neuronecrosis virus CP gene: The NNV-CP gene sequence of the virus was analyzed and the primers NNV-CP-F and NNV-CP-R were designed to amplify the cDNA of the NNV virus as a template. The homologous sequence and restriction site of the Pshuttle-CMV-ECMV vector were added to NNV-CP-F and NNV-CP-R respectively; (2) Construction of Pshuttle-CMV-ECMV vector carrying NNV-CP: The cloned NNV-CP gene was cloned into the Pshuttle-CMV-ECMV vector by homologous recombination technology to construct a Pshuttle-CMV-ECMV vector carrying the NNV-CP gene, named Pshuttle-CMV-ECMV / NNV-CP; (3) Obtaining recombinant adenovirus plasmid: homologously recombining the obtained recombinant adenovirus vector Pshuttle-CMV-ECMV / NNV-CP with the PAd-easy-1 vector in Escherichia coli BJ5183 to construct a recombinant adenovirus plasmid; (4) Screening and obtaining recombinant adenovirus: The screened recombinant adenovirus positive plasmid was digested with Pac Ⅰ and used to transfect HEK293 cells to obtain recombinant adenovirus carrying the NNV-CP gene; (5) Preparation of recombinant adenovirus vaccine for viral neuropathy: After the harvested recombinant adenovirus carrying the NNV-CP gene is subcultured, the expression of the target antigen protein is detected. The recombinant adenovirus that can express the protein after detection is inoculated into HEK293 cells for multiple subcultures, repeated freezing and thawing, and the virus titer is measured. The high-titer recombinant adenovirus is used to prepare the recombinant adenovirus vaccine for viral neuropathy.

3. The method for preparing the recombinant adenovirus vaccine for fish viral neuronecrosis according to claim 2, characterized in that: The nucleotide sequence of the primer NNV-CP-F is shown in SEQ ID NO:

2.

4. The method for preparing the recombinant adenovirus vaccine for fish viral neuronecrosis according to claim 2, characterized in that: The nucleotide sequence of the primer NNV-CP-R is shown in SEQ ID NO:

3.

5. The method for preparing the recombinant adenovirus vaccine for fish viral neuronecrosis according to claim 2, characterized in that: The high titer of the recombinant adenovirus should be greater than 1×10 8 mL -1 TCID50.

6. The method for preparing the recombinant adenovirus vaccine for fish viral neuronecrosis according to claim 2, characterized in that: In step (5), 1 ml of the high-titer recombinant adenovirus is taken to infect cultured HEK293 cells. After 72 hours of infection, the HEK293 cells and the supernatant are repeatedly frozen and thawed three times, and then centrifuged at 3000-4000 rpm / min for 8-10 minutes to obtain the fish rhabdovirus recombinant adenovirus vaccine.

7. Use of the fish viral neuronecrosis recombinant adenovirus vaccine as claimed in claim 1 for the preparation of a therapeutic drug for viral neuronecrosis caused by fish neuronecrosis virus.

8. Use of the fish viral neuronecrosis recombinant adenovirus vaccine as claimed in claim 1 for preparing an immersion preparation against fish neuronecrosis virus.

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