Fish rhabdovirus recombinant adenovirus vector vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a fish rhabdovirus recombinant adenovirus vector vaccine as well as a preparation method and application thereof. The replication-defective recombinant adenovirus vector is utilized to prepare the recombinant adenovirus vaccine carrying the M gene of the SCRV virus. The preparation method comprises the following steps: cloning the M gene of the mandarin fish rhabdovirus, constructing a Pshuttle-CMV-ECMV vector with a target gene SCRV-M, obtaining an SCRV-M recombinant adenovirus plasmid, screening to obtain the SCRV-M recombinant adenovirus, and preparing the SCRV-M recombinant adenovirus vaccine. The prepared SCRV-M recombinant adenovirus vaccine can be used for immunizing and preventing SCRV virus infection in a juvenile fish stage in a soaking manner, and the SCRV-M recombinant adenovirus vaccine has a wide application prospect in the aspect of prevention and control of fish rhabdovirus after being subjected to an animal safety test.

Description

Technical Field

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

[0002] SCRV is a negative-strand RNA virus belonging to the Rhabdoviridae family and the Perhabdovirus genus. It has a wide range of host infectivity and diversity, and can cause severe hemorrhagic sepsis in both freshwater and marine fish. Rhabdovirus has five major proteins, namely glycoprotein (G), matrix protein (M), nucleoprotein (N), phosphoprotein (NS) and RNA polymerase large protein (L). As one of the major pathogens in mandarin fish aquaculture, the presence of SCRV has caused huge losses to the aquaculture industry, and there is currently a lack of effective drug treatment and preventive measures. Traditional methods for the prevention and control of fish rhabdoviruses mainly rely on the development of inactivated or attenuated virus vaccines. Although inactivated vaccines have a certain protective effect, they require manual injection, which is cumbersome and not suitable for large-scale application. Although live attenuated vaccines are easily transmitted through water bodies, they have potential pathogenic risks, and direct release into water bodies may not comply with environmental control policies.

[0003] Adenovirus vaccines have become the first choice for gene expression and delivery of vaccines for various pathogens due to their high titer, high efficiency, and the fact that they will not integrate foreign genes into the host genome. Recombinant adenoviruses can effectively transfer target genes into a variety of cells and stimulate the body to produce effective and lasting specific humoral and cellular immune responses. In addition, recombinant adenoviruses can also infect a variety of teleost fish by immersion, showing extremely high infection efficiency and inducing effective mucosal immunity and T cell immune responses. In a variety of animal models, the mucosal immune response induced by adenovirus vectors is more effective than parenteral immunization, showing a long-lasting immune response. These characteristics make adenovirus vectors a promising vaccine candidate for preventing SCRV infection, and provide new ideas and methods for the development of immersion vaccines for fish rhabdovirus diseases. Summary of the invention

[0004] The present invention provides a fish rhabdovirus recombinant adenovirus vector vaccine and a preparation method and application thereof, which solves the problem that mandarin fish is susceptible to SCRV virus infection in culture and cannot be prevented by vaccine injection.

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

[0006] The preparation method of fish rhabdovirus recombinant adenovirus vector vaccine comprises the following steps: (1) Analyze the M gene of SCRV virus and design primers SCRV-MF and SCRV-MR to perform PCR amplification using SCRV virus cDNA as template. SCRV-MF and SCRV-MR were added with homologous sequences and restriction sites of Pshuttle-CMV-ECMV vector respectively; (2) Construction of a Pshuttle-CMV-ECMV vector carrying the target gene SCRV-M: The cloned SCRV-M gene was cloned into the Pshuttle-CMV-ECMV vector by homologous recombination technology to construct a Pshuttle-CMV-ECMV vector carrying the SCRV-M gene: Pshuttle-CMV-ECMV / SCRV-M; (3) Obtaining recombinant adenovirus plasmid: homologously recombining the obtained recombinant adenovirus vector Pshuttle-CMV-ECMV / SCRV-M 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 PacⅠ and used to transfect HEK293 cells to obtain recombinant adenovirus carrying the SCRV-M gene; (5) Preparation of fish rhabdovirus recombinant adenovirus vaccine: After the harvested recombinant adenovirus carrying the SCRV-M 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 fish rhabdovirus recombinant adenovirus vector vaccine.

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

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

[0009] Furthermore, the virus titer in step (5) must 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, followed by centrifugation at 3000-4000 rpm / min for 8-10 minutes to obtain the fish rhabdovirus recombinant adenovirus vector vaccine.

[0011] The invention discloses an application of a fish rhabdovirus recombinant adenovirus vector vaccine for preparing a medicine for treating hemorrhagic septicemia caused by mandarin fish rhabdovirus.

[0012] The invention discloses an application of a fish rhabdovirus recombinant adenovirus vector vaccine in the preparation of an immersion preparation against fish rhabdovirus.

[0013] Beneficial effects of the present invention: (1) The present invention is based on the principle that the M gene of SCRV virus is a key component of the viral genome, and the M protein is mainly involved in the assembly process of virus particles and has a key influence on the morphology and infectivity of the virus. After amplifying the M gene of mandarin fish rhabdovirus, it is connected to the Pshuttle-CMV-ECMV vector, and after PCR screening is positive, it is homologously recombined with the PAd-easy-1 backbone vector to construct a replication-deficient recombinant adenovirus plasmid. After transfecting cells with the plasmid, a replication-deficient recombinant adenovirus containing the target gene is obtained; the recombinant adenovirus after detection is inoculated into HEK293 cells for multiple passages, and a high-titer recombinant adenovirus is selected to prepare a SCRV-M recombinant adenovirus vaccine, which has high vaccine stability; (1) The SCRV-M recombinant adenovirus vaccine prepared by the present invention can be immunized by immersion, and can be used for immunization in the fish fry stage. The immune protection rate is as high as 80% 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.

[0014] (2) The results of animal safety tests on the SCRV-M recombinant adenovirus vaccine prepared by the present invention show that the vaccine is safer, more effective, and has no toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The following is a diagram of the construction process of the SCRV-M recombinant adenovirus vector of the present invention: A: Identification of SCRV-M gene PCR products: Lane 1, DNA Maker; Lane 2, SCRV-M gene PCR amplification products; Lane 3: blank control. B: PCR amplification and double enzyme digestion results of recombinant plasmid: Lane 1, DNA Maker; Lane 2, Sal I and XhoⅠ double enzyme digestion results; Lane 3, PCR amplification results; Lane 4, empty vector PCR amplification results. C: Identification of recombinant adenovirus vector: Lane 1, DNA Maker; Lane 2, Pac I digestion results of recombinant adenovirus plasmid; Lane 3, M gene PCR amplification results; Lane 4, blank control.

[0016] Figure 2 The green fluorescence expression results of the SCRV-M 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.

[0017] Figure 3 The Western-blot detection results of SCRV-M protein expression of the SCRV-M recombinant adenovirus of the present invention are as follows: Lane 1, the expression results of the recombinant adenovirus M 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.

[0018] Figure 4 This is a graph showing the survival rate of mandarin fish 7 days after being treated with the SCRV-M recombinant adenovirus vaccine of the present invention.

[0019] Figure 5 This is a schematic diagram of the effect of the SCRV-M recombinant adenovirus vaccine of the present invention in preventing and treating SCRV virus infection in mandarin fish. DETAILED DESCRIPTION

[0020] 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.

[0021] Human embryonic kidney 293 cells (HEK293 cells) adhered to the wall and grew in a cell culture medium 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 HEK293 cells; DMEM medium containing only 2% fetal bovine serum was used as the maintenance medium for HEK293 cells during the culture process.

[0022] The Siniperca chuatsi rhabdovirus (SCRV) selected in the present invention is stored in the National Aquatic Animal Pathogen Bank of the Ministry of Agriculture and Rural Affairs.

[0023] The DNA purification and recovery kit and the plasmid extraction kit in the embodiments of the present invention are both products of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0024] The Pshuttle-CMV-ECMV vector in the examples of the present invention was purchased from Haizeye Biotechnology Co., Ltd.

[0025] The high-fidelity enzyme premix in the examples of the present invention was purchased from Nanjing Novozyme Biotech Co., Ltd.

[0026] Example 1: Construction of SCRV-M recombinant adenovirus vector: (1) Cloning of the M gene of mandarin fish rhabdovirus: First, primers SCRV-MF and SCRV-MR were used to amplify the DNA template of SCRV-M using SCRV virus cDNA as a template. The sequence of SCRV-MF is SEQ ID NO: 2: 5'-ATTTGCGGCCGCGCCACCATGCCTCTGTTTAAGAAGAG-3' The sequence of primer SCRV-MR is SEQ ID NO: 3: (5'-CCGCTCGAGATGCCAGCTATGACCAG-3'), The specific steps are as follows: SCRV-MF 1μL (10μM), SCRV-MR 1μL (10μM), SCRV virus cDNA 1μL, high-fidelity enzyme premix 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 SCRV-M gene PCR. After obtaining a single SCRV-M gene target fragment, the above PCR product was purified using a DNA purification recovery kit; the results are as follows Figure 1 As shown in A.

[0027] Depend on Figure 1 As shown in the results, the SCRV-M gene DNA template was successfully amplified; (2) Construction of Pshuttle-CMV-ECMV vector carrying target gene (SCRV-M): The purified SCRV-M 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 SCRV-M gene. The SCRV-M 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 12-16 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 bacterial solution after expansion culture to confirm the insertion of the SCRV-M target gene. The bacterial solution 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 and sequenced to verify the sequence correctness of the recombinant plasmid; the results are as follows Figure 1 As shown in B.

[0028] like Figure 1 As shown in B, the SCRV-M fragment has been successfully homologously recombined into the Pshuttle-CMV-EGFP vector.

[0029] (3) Obtain Pshuttle-CMV-EGFP / SCRV-M recombinant adenovirus plasmid: First, the Pshuttle-CMV-EGFP / SCRV-M vector was digested with Pme I to obtain a linearized vector. Subsequently, the linearized Pshuttle-CMV-EGFP / SCRV-M vector was transformed into BJ5183 competent cells, which already contained the PAd-easy-1 helper 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 coated on an LB plate containing 100 μg / mL kanamycin for screening. The PCR products were detected by 0.8% agarose gel electrophoresis to confirm the insertion of the target gene. After a large number of positive products were cultured, plasmid DNA was extracted and digested with Pac I restriction endonuclease to verify the correctness of the recombinant adenovirus plasmid. The screened positive plasmids were further transformed into Stbl3 competent cells by heat shock to obtain a large amount of high-quality plasmid DNA. The selected positive clones were digested again 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 as follows Figure 1 As shown in C.

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

[0031] (4) Screening to obtain SCRV-M 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 the intracellular GFP expression and cytopathic effect (ME). 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] like Figure 2 The results showed that a large amount of GFP fluorescence could be observed in HEK293 cells transfected with adenovirus vaccine plasmid SCRV-M 48 hours after transfection, while no GFP fluorescence was observed in negative control HEK293 cells, indicating that the adenovirus vaccine plasmid SCRV-M was successfully constructed.

[0034] (5) Preparation of fish rhabdovirus recombinant adenovirus vaccine: HEK293 cells in the logarithmic growth phase were obtained and digested with trypsin 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%, 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 is reached, the recombinant adenovirus stock solution is used to prepare a recombinant adenovirus vaccine of rhabdovirus: the specific steps are as follows: HEK293 cells in the logarithmic growth phase were obtained and digested with trypsin 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, and 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 solution was added. After culturing for 72 hours, the HEK293 cells and the supernatant were repeatedly frozen and thawed 3 times, and then centrifuged at 4000 rpm / min for 10 minutes, and the supernatant was taken. The supernatant was the rhabdovirus recombinant adenovirus vaccine.

[0036] The results are as follows Figure 3 shown.

[0037] like Figure 3 The results showed that HEK293 cells infected with SCRV-M recombinant adenovirus vaccine could stably express SCRV-M protein, while HEK293 cells infected with recombinant empty vector adenovirus and blank HEK293 cells did not express SCRV-M protein. This indicates that SCRV-M recombinant adenovirus vaccine can successfully express the target protein SCRV-M, and SCRV-M recombinant adenovirus vaccine is successfully constructed.

[0038] Example 2: Safety and efficacy verification of SCRV-M recombinant adenovirus vaccine: In order to comprehensively evaluate the safety of the recombinant adenovirus vaccine in the present invention for preventing rhabdovirus 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. The test selected 5g mandarin fish as the experimental animal, and the titer of the recombinant adenovirus vaccine used was 1×10 9 mL -1 TCID50.

[0039] In the experimental design, the first group of mandarin fish was 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 mandarin fish was inoculated with 0.1 mL of the recombinant adenovirus vaccine per tail by injection; the third group of mandarin fish served as a control group that was not vaccinated. During the experiment, all experimental groups of mandarin fish 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 fish in each group were regularly measured and recorded within 60 days after vaccination. The results are as follows Figure 4 It is knowable.

[0040] according to Figure 4The results shown in the figure show that, while the mandarin fish in the control group were in good growth condition, no fish deaths were observed in the first two groups vaccinated within 7 days, and no clinical adverse reactions were found. Moreover, compared with the control group, there was no significant difference in the length and weight of the fish in the vaccinated group after 60 days. These results show that the SCRV-M recombinant adenovirus vaccine has a high safety profile, providing a scientific basis for its clinical application.

[0041] Example 3: Application of SCRV-M recombinant adenovirus vaccine: Mandarin fish weighing about 10 g were selected and vaccinated by immersion. 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 vaccination 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 mandarin fish were intraperitoneally injected with 20 μL of 1×10 5 TCID50 / mL of SCRV virus. During the subsequent observation period, the survival and death of each group of mandarin fish were recorded every day, and a survival curve was drawn based on these data to evaluate the protective effect of the vaccine. Figure 5 shown.

[0042] according to Figure 5 The experimental results shown in the figure show that the mortality rate of mandarin fish vaccinated with SCRV-M recombinant adenovirus vaccine was about 20% during the 20-day observation period. In contrast, the mortality rate of mandarin fish in the negative control group that was not vaccinated reached 70% during the same period of time. This significant difference indicates that the SCRV-M recombinant adenovirus vaccine vaccinated by immersion has an 80% protective efficacy against SCRV virus infection in mandarin fish.

[0043] 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 fish rhabdovirus recombinant adenovirus vector vaccine, characterized in that: The antigen protein of the recombinant adenovirus vector vaccine contains the M gene of the SCRV virus, and the nucleotide sequence of the antigen of the recombinant adenovirus vector vaccine is shown in SEQ ID NO:

1.

2. The method for preparing the fish rhabdovirus recombinant adenovirus vector vaccine according to claim 1, characterized in that: The steps include: (1) Analyze the M gene of SCRV virus and design primers SCRV-MF and SCRV-MR to perform PCR amplification using SCRV virus cDNA as template. SCRV-MF and SCRV-MR were added with homologous sequences and restriction sites of Pshuttle-CMV-ECMV vector respectively; (2) Construction of a Pshuttle-CMV-ECMV vector carrying the target gene SCRV-M: The cloned SCRV-M gene was cloned into the Pshuttle-CMV-ECMV vector by homologous recombination technology to construct a Pshuttle-CMV-ECMV vector carrying the SCRV-M gene: Pshuttle-CMV-ECMV / SCRV-M; (3) Obtaining recombinant adenovirus plasmid: homologously recombining the obtained recombinant adenovirus vector Pshuttle-CMV-ECMV / SCRV-M 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 PacⅠ and used to transfect HEK293 cells to obtain recombinant adenovirus carrying the SCRV-M gene; (5) Preparation of fish rhabdovirus recombinant adenovirus vector vaccine: After the harvested recombinant adenovirus carrying the SCRV-M gene is passaged, 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 passages, repeated freezing and thawing, and the virus titer is measured. The high-titer recombinant adenovirus is used to prepare the fish rhabdovirus recombinant adenovirus vector vaccine.

3. The method for preparing the fish rhabdovirus recombinant adenovirus vector vaccine according to claim 2, characterized in that: The nucleotide sequence of the primer SCRV-MF is shown in SEQ ID NO:2, and the nucleotide sequence of the primer SCRV-MR is shown in SEQ ID NO:

3.

4. The method for preparing the fish rhabdovirus recombinant adenovirus vector vaccine according to claim 2, characterized in that: The virus titer in step (5) must be greater than 1×10 8 mL -1 TCID50.

5. The method for preparing the fish rhabdovirus recombinant adenovirus vector vaccine 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 vector vaccine.

6. Use of the fish rhabdovirus recombinant adenovirus vector vaccine as claimed in claim 1 in the preparation of a drug for treating hemorrhagic septicemia caused by mandarin fish rhabdovirus.

7. Use of the fish rhabdovirus recombinant adenovirus vector vaccine as claimed in claim 1 in the preparation of an anti-fish rhabdovirus soaking preparation.

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