Preparation method and application of larimichthys crocea iridovirus disease recombinant live vector vaccine

Abstract

The invention relates to the field of aquaculture, in particular to a preparation method and application of a larimichthys crocea iridovirus recombinant live vector vaccine. According to the invention, replication-deficient type 5 adenovirus is selected as a target gene expression vector, a recombinant adenovirus vector comprising main capsid protein of iridovirus is constructed, recombinant virus is obtained, and the safe and effective large yellow croaker iridovirus disease recombinant live vector vaccine is created. According to the vaccine, the larimichthys crocea is immunized in a soaking or bait feeding mode, the CPM of the larimichthys crocea immunized by the vaccine is 12.0%, and the CPM of the fish without being immunized by the vaccine is 62.0%. In addition, the RPS of the inoculated fish and the RPS of the empty carrier control group fish are 80.65% and 9.68% respectively. The method is at a leading level in the field of pseudosciaena crocea biomedicine at present, and creativity is remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture and recombinant live vector vaccine creation, specifically, a method for preparing a recombinant live vector vaccine for large yellow croaker iridovirus disease by using a replication-defective recombinant adenovirus vector, and particularly to a method for preparing a recombinant live vector vaccine for large yellow croaker iridovirus disease by using a recombinant adenovirus vector to express the major capsid protein of large yellow croaker iridovirus. Background Art

[0002] Large yellow croaker (Larimichthys crocea) belongs to the class of bony fishes, order of Perciformes, family of Sciaenidae, and genus of Pseudosciaena. It is commonly known as yellow croaker, yellow croaker, cucumber fish, etc. It is an important marine aquaculture fish.

[0003] While the large yellow croaker aquaculture industry is rapidly developing, it is also facing challenges such as deteriorating genetic resources, rising feed costs, and frequent disease outbreaks, with large yellow croaker diseases being particularly prominent. The main diseases prevalent in large yellow croaker include viral diseases such as iridovirus disease, bacterial diseases such as iridovirus disease, and parasitic diseases such as Cryptocaryon irritans. Large yellow croaker iridovirus disease is one of the most serious viral diseases, with a high incidence rate among fry and a mortality rate as high as 50%. This extremely high mortality rate has caused severe economic losses to the large yellow croaker aquaculture industry.

[0004] Large yellow croaker iridovirus disease primarily infects juvenile yellow croaker. Initially, infected fish remain intact, swim slowly, and eat less. As the disease progresses, the gills become pale, the spleen and kidneys become enlarged, and the liver becomes congested. The fish refuse to eat and eventually die. The causative agent of large yellow croaker iridovirus disease is Large yellow croaker iridovirus (LYCIV), a large, icosahedral cytoplasmic DNA virus belonging to the Iridoviridae family and the genus Cytosoma Iridovirus. The virus primarily occurs during the summer heatwave, with an optimal water temperature of 27°C to 30°C.

[0005] Vaccination is one of the best means of controlling outbreaks of major aquatic animal diseases. Currently, there are no reports on commercial vaccines, drugs, or other preventive agents for yellow croaker iridovirus disease. In actual yellow croaker production, the treatment for iridovirus disease is starvation therapy. Therefore, outbreaks of iridovirus disease remain a significant challenge in the healthy and sustainable large-scale aquaculture of yellow croaker. Therefore, developing a more effective and convenient vaccine for the prevention and control of iridovirus disease in yellow croaker is crucial to reduce economic losses for yellow croaker farmers. Summary of the Invention

[0006] The purpose of the present invention is to solve the technical problem of the outbreak of iridovirus disease in the above-mentioned large yellow croaker breeding process. In order to solve the problem of iridovirus infection in the large yellow croaker breeding process, combined with existing research, a preparation method and application of a live vector vaccine for preventing iridovirus disease in large yellow croaker are provided, which is safe and can effectively prevent iridovirus disease in large yellow croaker.

[0007] The first aspect of the present invention provides a method for preparing a recombinant live vector vaccine for large yellow croaker iridovirus disease, comprising the following steps: amplifying the MCP (Major capsid protein, MCP) gene of large yellow croaker iridovirus, constructing it into the pAd-Track-CMV shuttle vector, and then recombining it with the pAd-Easy-1 backbone vector in Escherichia coli to construct an MCP gene recombinant adenovirus plasmid, linearizing the recombinant adenovirus plasmid and transfecting it into a 293T cell line to obtain a replication-defective recombinant adenovirus; inoculating the positive recombinant adenovirus into 293 suspension cells for multiple passages, and selecting a stable high-titer recombinant adenovirus to prepare a recombinant live vector vaccine for large yellow croaker iridovirus disease.

[0008] Furthermore, the nucleotide sequence of the MCP gene is shown in SEQ ID NO. 1, and the nucleotide sequences of the primers used to amplify the MCP gene are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively. The forward primer for MCP gene amplification incorporates a sequence homologous to the shuttle vector and a Bgl II restriction enzyme sequence, and is shown in SEQ ID NO. 2; the reverse primer incorporates a sequence homologous to the shuttle vector and a Not I restriction enzyme sequence, and is shown in SEQ ID NO. 3. The nucleotide sequences of the primers used for PCR screening are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0009] Furthermore, the method for preparing the recombinant live vector vaccine for large yellow croaker iridovirus disease comprises the following steps:

[0010] (A) Amplification of the MCP gene

[0011] The MCP gene was amplified from the genome of the large yellow croaker iridovirus. The upstream primer and downstream primer of the MCP gene were added with homologous sequences of the pAd-Track-CMV shuttle vector and Bgl II and Not I restriction sites, respectively. The nucleotide sequence of the MCP gene is shown in SEQ ID NO. 1, and the nucleotide sequences of the primers used to amplify the MCP gene are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0012] (B) Rapid ligation of the MCP gene with the pAd-Track-CMV shuttle vector

[0013] The cloned MCP gene was connected to the pAd-Track-CMV shuttle vector using rapid fusion technology to obtain a recombinant shuttle vector containing the MCP gene;

[0014] (C) Homologous recombination between the pAd-Track-CMV shuttle vector and the pAd-Easy-1 backbone vector

[0015] The pAd-Track-CMV shuttle vector carrying the MCP gene was recombined with the pAd-Easy-1 backbone vector in Escherichia coli to obtain a recombinant adenovirus plasmid named Adv-MCP.

[0016] (D) Recombinant adenovirus packaging and preparation

[0017] The recombinant plasmid Adv-MCP in step (C) was linearized by Pac Ⅰ digestion and then transfected into 293T cell line for virus packaging and preparation to obtain recombinant adenovirus;

[0018] (E) Detection of recombinant virus antigen protein expression level

[0019] Western-blot method was used to detect the expression of MCP protein to confirm that the constructed recombinant adenovirus could express the target protein;

[0020] (F) Preparation of a recombinant live vector vaccine for yellow croaker iridovirus disease

[0021] After multiple passages, the recombinant adenovirus that can express the target protein is harvested and the recombinant adenovirus with a stable and high virus titer is harvested. After repeated freezing and thawing, centrifugation, and re-proportioning, the recombinant live vector vaccine for yellow croaker iridovirus disease is prepared.

[0022] Furthermore, the step (B) of rapidly connecting the MCP gene with the pAd-Track-CMV shuttle vector comprises the following steps:

[0023] (b1) Gene ligation vector: Double-digest the adenoviral shuttle vector with the nucleases Bgl II and Not I. After electrophoresis on 0.8% agarose gel, recover and purify the target fragment according to the kit instructions. Ligate the multiple fragments to the adenoviral shuttle vector using the Rapid Cloning Kit. The kit is used to create a reaction mixture containing 40 ng of positive PCR multi-fragment product, 20 ng of linearized pAd-Track-CMV, 1 µL of Rapid Fusion Enzyme, 2 µL of 10x Fusion Enzyme Buffer, and ddH2O to a total reaction volume of 20 µL. Reaction conditions: Incubate in a water bath at 37°C for 30 min.

[0024] (b2) Transformation of ligation product: Immediately after the reaction, transform 10 µL of the ligation product into 40 µL of competent cells. Spread the transformed bacteria onto LB plates containing 50 µg / mL kanamycin and incubate at 37°C for 16–20 h. Pick 15–20 colonies from the plate and inoculate them into 5 mL of LB broth containing 50 µg / mL kanamycin. Incubate at 220 rpm at 37°C for 16–20 h.

[0025] (b3) Preliminary PCR screening of positive clones: Plasmids were extracted according to the instructions of the plasmid extraction kit and identified by PCR amplification (forward and reverse primers are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively). The expected total size of the MCP gene ligation product was 1394 bp. PCR products were electrophoresed on a 0.8% agarose gel and visualized under ultraviolet light. An empty vector control was also included. PCR products were stored at -20°C until needed.

[0026] (b4) Double enzyme digestion: Plasmids identified as positive by PCR were digested with Bgl II and Not I restriction enzymes. The results were then analyzed by electrophoresis on a 0.8% agarose gel and imaged under UV light. The pAd-Track-CMV vector plasmid was used as a negative control. Positive products were sent to a sequencing company for sequencing.

[0027] Furthermore, the step (C) comprises the following steps:

[0028] (c1) Dual vector ligation: The pAd-Track-CMV shuttle vector was linearized by Pme I digestion and then electroporated into competent cells carrying the pAd-Easy-1 backbone vector.

[0029] (c2) Identification of positive plasmids: Plasmids are extracted from the cultured bacterial suspension. After PCR amplification, the recombinant viral plasmids are identified by electrophoresis on a 0.8% agarose gel. Suspected positive plasmids are identified by digestion with the endonuclease Pac I (NEB) at 37°C for 2-4 hours.

[0030] (c3) High-copy plasmid construction: The positive recombinant plasmid was electroporated into competent Escherichia coli DH10B cells. After Pac I digestion, the Ori and kanamycin resistance elements on the vector were removed and the DNA was recovered for transfection.

[0031] Furthermore, the step (D) comprises the following steps:

[0032] (d1) Host cell preparation: 24 h before transfection, 293T cells with uniform morphology and good growth status were cultured at 1.0×10 6 mL -1The concentration of inoculation was 25 cm 2 The cells were cultured in a cell culture flask. Transfection was performed when the cells were approximately 50-70% adherent.

[0033] (D2) Transfection: Add Advanced Transfection Reagent and linearized DNA to DMEM to prepare a culture suspension and incubate at room temperature for 30 minutes. Add the mixture to the cells and culture for 8 hours. Continue culturing in DMEM supplemented with 2% serum.

[0034] (d3) Harvesting the recombinant virus: 2-8 days after transfection, monitor the expression of green fluorescent protein (GFP) in the cells and harvest the cell culture medium when the fluorescence reaches the brightest.

[0035] Furthermore, the step (E) comprises the following steps:

[0036] (e1) Sample preparation: Harvest virus-infected 293T cells, freeze-thaw repeatedly several times, and then sonicate to fully lyse the cells. Proteins were extracted from the lysate using RIPA buffer. The collected sample was centrifuged at 12,000 rpm for 5 minutes at 4°C. The supernatant was then transferred to a new tube and loaded with protein loading buffer. Protein samples were prepared by incubating at 95°C for 15 minutes.

[0037] (e2) Western blot analysis: Protein samples were electrophoresed on an SDS-PAGE gel at 80 V for 60 minutes. The voltage was then adjusted to 120 V until the end of the electrophoresis. β-actin was used as an internal control, and the protein samples were electrotransferred to a PVDF membrane at 300 mA for 90 minutes. After transfer, the membrane was blocked with bovine serum albumin (50 μg / L) and incubated with a mouse anti-His tag antibody (HRP-conjugated) for 2 hours. Protein detection was performed by adding a prepared developer for chemiluminescence reaction.

[0038] Furthermore, the step (F) comprises the following steps:

[0039] (f1) Virus collection: The harvested virus liquid seed virus is repeatedly frozen and thawed 2-3 times, and then centrifuged to remove cell debris. Centrifuge at 5000 rpm for 5 minutes at 4°C, and then collect the supernatant to determine the virus titer. The virus with the high titer is retained as seed virus for batch preparation of live vector vaccine.

[0040] (f2) Screening for the optimal cell line: The virus seed is inoculated into various adherent and suspension cell lines, and the cytopathic effect (CPE) of the virus is analyzed and its titer is measured. On this basis, the optimal conditions for viral replication are screened and the lowest-cost preparation method is calculated.

[0041] (f3) Vaccine preparation: Recombinant viruses were prepared in batches according to the virus preparation method with the lowest cost and best culture. The harvested virus stock was frozen and thawed 2-3 times, and then centrifuged to remove cell debris. The recombinant viruses were then enriched by ultracentrifugation and resuspended in PBS to prepare the recombinant live vector vaccine for yellow croaker iridovirus disease.

[0042] The second aspect of the present invention provides a recombinant live vector vaccine for large yellow croaker iridovirus disease, which is prepared using the method described above.

[0043] In the safety test, the recombinant live vector vaccine for yellow croaker iridovirus disease was administered to yellow croaker by immersion and intraperitoneal injection, respectively. Within 14 days after immunization, no fish died in the two experimental groups (immersion and injection), and the blank control group, nor did any local or systemic clinical adverse reactions occur. 60 days after vaccination, there was no significant difference in the length and weight of the fish in the two experimental groups (immersion and injection), compared with the control group. This test shows that the recombinant live vector vaccine for yellow croaker iridovirus disease prepared by the present invention is safe, providing an experimental basis for the next step of the clinical application of this vaccine in yellow croaker.

[0044] The third aspect of the present invention provides a use of the above-mentioned recombinant live vector vaccine for large yellow croaker iridovirus disease in the preparation of an agent for preventing large yellow croaker iridovirus disease.

[0045] The vaccine was administered to large yellow croaker by immersion immunization, followed by infection with the iridovirus by immersion. Compared to the unvaccinated control group, the mortality rate of the vaccinated fish was significantly lower. The cumulative percent mortality (CPM) of the vaccinated fish was 12.0%, compared to 62.0% in the control group. Furthermore, the relative percent survival (RPS) of the vaccinated fish and the empty vector control group was 80.65% and 9.68%, respectively. These results demonstrate that the vaccine prepared in this invention is effective in preventing iridovirus infection in large yellow croaker.

[0046] The beneficial effects of the present invention are as follows:

[0047] The present invention selects type 5 replication-deficient adenovirus as the target gene expression vector, constructs a recombinant adenovirus vector including the major capsid protein of the iridovirus and obtains the recombinant virus, thereby creating a safe and effective recombinant live vector vaccine for large yellow croaker iridovirus disease.

[0048] This invention addresses the urgent challenge of large yellow croaker iridovirus disease, a major issue in the large yellow croaker aquaculture industry. Through numerous experiments and in conjunction with the current state of research on large yellow croaker iridovirus vaccines, the present invention ultimately developed its overall technical solution. Using a replication-defective recombinant adenovirus as a target gene expression vector, the present invention constructs a recombinant adenovirus vector capable of expressing the large yellow croaker iridovirus MCP protein, screens for recombinant adenoviruses, and optimizes the virus preparation method with the lowest cost and best culture conditions to produce a recombinant live vector vaccine for large yellow croaker iridovirus disease. This vaccine utilizes an immersion method to immunize large yellow croaker for the prevention and control of large yellow croaker iridovirus disease, demonstrating significant innovation.

[0049] In terms of safety, the results of animal tests of the present invention showed that no dead fish were found in the test groups within 14 days after the vaccine was inoculated with large yellow croaker using different methods, and no clinical adverse reactions occurred. Moreover, 60 days after inoculation, there was no significant difference in the length and weight of the fish compared with the unvaccinated control group.

[0050] In terms of vaccine efficacy, economic, and social benefits, the mortality rate of large yellow croaker infected with iridovirus was reduced from over 62.0% to 12.0% after vaccination compared to the unvaccinated control group. Furthermore, the RPS of vaccinated fish and the empty vector control group were 80.65% and 9.68%, respectively. This vaccine is currently at the forefront of large yellow croaker biomedicine and represents a significant innovation. If commercially available, it could reduce annual economic losses for fishermen by over 100 million yuan, generate over 100 million yuan in economic benefits for the vaccine manufacturer, and create jobs for thousands of people, thus generating significant social benefits.

[0051] In summary, compared to other vaccines or effective prevention and treatment methods for large yellow croaker iridovirus disease, the recombinant live vector vaccine for large yellow croaker iridovirus disease prepared by the present invention is safer, more effective, and has no toxic side effects. The immunization method is relatively simple to perform clinically and can effectively reduce the mortality rate of large yellow croaker infected with iridovirus, thus possessing extremely broad application prospects in the prevention and control of large yellow croaker iridovirus disease. The development and research of the recombinant live vector vaccine for large yellow croaker iridovirus disease of the present invention aligns with the requirements for the green and healthy development of the aquaculture industry, complies with industrial policies for veterinary drug development, and meets the safety requirements for aquatic animals and related foods. It is beneficial to the healthy and rapid development of the aquaculture industry and has important practical significance for the production of green and pollution-free aquatic products. It also meets the needs of the modern development of new veterinary drugs for aquatic animals with independent intellectual property rights. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Results of constructing a recombinant adenovirus expressing the MCP gene: A. PCR amplification product of the MCP gene; M. 5000 bp DNA marker; 1. PCR amplification of the MCP gene; B. PCR amplification product and double enzyme digestion results of the recombinant plasmid; M. 10000 bp DNA marker; 1. Double enzyme digestion of the recombinant plasmid with Bgl II and Not I; 2. PCR amplification product of the recombinant plasmid; C. Identification of the recombinant adenovirus vector; M. 15000 bp DNA marker; 1, 2: Recombinant adenovirus plasmid digested with Pac I; 3. PCR amplification of the MCP gene.

[0053] Figure 2 Western blot results of recombinant adenovirus MCP protein expression: 1. MCP protein expression of recombinant adenovirus harvested from 293T cells; 2. Recombinant empty vector control virus harvested from 293T cells; 3. Control group 293T cells.

[0054] Figure 3 Green fluorescence expression results 5 days after recombinant adenovirus transfection: A. Green fluorescent protein (GFP) expression in 293T cells infected with recombinant adenovirus 24 hours later; B. 293T cells and control group (fluorescence background); C. 293T cells and control group (bright field).

[0055] Figure 4 Cumulative mortality (CPM) of large yellow croaker infected with iridovirus within 30 days after vaccination 15 days after vaccination: Asterisks (*) indicate significant differences in gene expression levels (P < 0.05). DETAILED DESCRIPTION

[0056] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0057] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0058] Example 1: Preparation method of recombinant live vector vaccine for large yellow croaker iridovirus disease

[0059] (1) Amplification of target gene (MCP gene)

[0060] Iridocidal virus DNA was extracted using a DNA extraction kit. MCP gene primers were used to amplify the target gene cDNA sample (GenBank accession number: AY779031.1). The primer sequences used to amplify the MCP gene are shown in SEQ ID NOs. 2 and 3, respectively. Bgl II and Not I restriction sites were added to the upstream and downstream primers, respectively. The corresponding restriction sites are underlined in the sequences. The primers were synthesized by Wuhan Jinkairui Biotechnology Co., Ltd., and the expected amplified sequence length of the MCP gene was 1394 bp. The PCR amplification system consisted of 1× PrimeSTAR GXL buffer, a 200 µM dNTP mix, 2.5 units of PrimeSTAR GXL DNA polymerase, 2 µL of sample cDNA, and primers at 0.2-0.3 µM concentrations. ddH2O was added to bring the total volume to 50 µL. Amplification was performed in a PCR instrument for 31 cycles. Amplification parameters were: initial denaturation at 94°C for 2 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 2 minutes, and a final extension at 72°C for 10 minutes. 10 µL of product was electrophoresed on a 1.5% agarose gel and detected under ultraviolet light. Positive products were identified and sequenced and aligned with the target sequence.

[0061] (2) Rapid ligation of MCP gene and pAd-Track-CMV vector

[0062] The correctly identified MCP gene was added to the Rapid Cloning Reagent master reaction. The adenoviral shuttle vector was double-digested with endonucleases Bgl II and Not I. The target fragment was recovered and purified following 0.8% agarose gel electrophoresis according to the kit instructions. Ligation of the multiple fragments into the adenoviral shuttle vector was performed using the Rapid Cloning Kit. The kit was used to create a reaction mixture containing 40 ng of positive PCR multiple fragment product, 20 ng of linearized pAd-Track-CMV, 1 µL of Rapid Fusion Enzyme, 2 µL of 10x Fusion Enzyme Buffer, and ddH2O to a total reaction volume of 20 µL. Reaction conditions: Incubate in a water bath at 37°C for 30 min.

[0063] Immediately after the reaction, 10 µL of the ligation product was transformed into 40 µL of competent cells. The transformation steps were as follows: First, the competent cells were incubated on ice for 30 minutes, then placed in a 42°C water bath for 45 seconds. The mixture was then immediately incubated at 220 rpm at 37°C for 1 hour. The mixture was then spread onto LB plates containing 50 µg / mL kanamycin and incubated at 37°C for 16-20 hours. Fifteen to 20 colonies were then picked from the plates and inoculated into 5 mL of LB liquid medium containing 50 µg / mL kanamycin and incubated at 220 rpm at 37°C for 16-20 hours. The plasmid was extracted according to the instructions of the plasmid extraction kit and identified by PCR amplification (forward and reverse primers are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively). The expected total size of the MCP gene ligation product was 1394 bp. The PCR reaction system consisted of 2 µL of plasmid, 1 µL of DNA polymerase, 10 µL of 5× DNA polymerase buffer, 4 µL of dNTP mix, and 2.0 µL each of upstream and downstream primers (pre-diluted to 10 µmol / L). Add ddH₂O to a total of 50 µL. Reaction conditions included initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 94°C for 10 s, annealing at 62°C for 5 min, and extension at 72°C for 2 min. The final extension was at 70°C for 10 min. PCR products were electrophoresed on a 0.8% agarose gel and visualized under UV light. Empty vector was also included as a control. PCR products were stored at -20°C until ready for use.

[0064] Plasmids identified as positive by PCR were digested with Bgl II and Not I restriction enzymes. The results were then analyzed by electrophoresis on a 0.8% agarose gel and visualized under UV light. The pAd-Track-CMV vector plasmid served as a negative control. Positive products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0065] (3) Homologous recombination between the pAd-Track-CMV shuttle vector and the pAd-Easy-1 backbone vector

[0066] The pAd-Track-CMV shuttle vector was electroporated into competent cells harboring the pAd-Easy-1 backbone vector and cultured in 5 mL of LB liquid medium containing 50 μg / mL kanamycin at 220 rpm at 37°C for 16–20 h. Plasmids were extracted from the culture medium and amplified by PCR. Recombinant adenoviral plasmids were identified by electrophoresis on a 0.8% agarose gel and imaged under UV light. Suspected positive plasmids were confirmed by digestion with the restriction endonuclease Pac I (NEB) at 37°C for 2–4 h. The reaction system contained 1 μg of positive adenoviral plasmid, 5 μL of 10× buffer, 1 μL of the restriction endonuclease Pac I, and ddH2O to a total reaction volume of 50 μL. Positive recombinant adenoviral plasmids were then electroporated into competent Escherichia coli DH10B cells. After digestion with Pac I, the Ori and kanamycin resistance genes on the vector were removed, and the DNA was recovered for transfection. The Pac I reaction system contains 1 µg of positive adenoviral plasmid, 5 µL of 10× buffer, 1 µL of restriction endonuclease PacI, and ddH2O to a total reaction volume of 50 µL. Pac I digestion is performed at 37°C for 2-4 hours, and the digested fragments are recovered for later use.

[0067] (4) Recombinant adenovirus packaging and screening

[0068] The transfection steps of 293T cells with recombinant adenovirus plasmids were as follows: 24 h before transfection, 293T cells with uniform morphology and good growth status were cultured at 1.0×10 6 mL -1 The concentration of inoculation was 25 cm 2 The cells were cultured in a cell culture flask. When the cells were about 50-70% adherent, transfection was performed. Advanced transfection reagent and linearized DNA were added to DMEM to make a culture suspension and incubated at room temperature for 30 minutes. The mixture was then added to the cells and cultured for 8 hours. Then, 2% serum DMEM maintenance medium was added to continue the culture. 2-8 days after transfection, the expression of GFP in the cells was monitored, and the remaining cell culture fluid was harvested when the fluorescence was brightest. Under a fluorescence microscope, the transfected virus was passaged and proliferated according to the expression level of GFP. 293T cells transfected with a recombinant empty vector control virus were used as a negative control. After the recombinant virus was propagated in 293T cells for 5 generations, stable lesions (GFP covered the cells) appeared in 24-36 hours, and the harvested virus fluid was used to detect whether the target representative was expressed.

[0069] (5) Detection of expression levels of recombinant viral antigen proteins

[0070] Virus-infected 293T cells were harvested, frozen and thawed several times, and then sonicated to fully lyse the cells. Proteins were extracted from the lysate using RIPA buffer. The collected samples were centrifuged at 12,000 rpm for 5 minutes at 4°C. The supernatant was then transferred to a new tube and loaded with protein loading buffer. Protein samples were prepared at 95°C for 15 minutes. Protein samples were electrophoresed on SDS-PAGE gels at 80 V for 60 minutes, after which the voltage was adjusted to 120 V until the end of the run. β-actin was used as a loading control, and the protein samples were electrotransferred to a PVDF membrane at 300 mA for 90 minutes. After transfer, the membrane was blocked with bovine serum albumin (50 g / L) for 1 hour and washed three times with PBST buffer (10 minutes each). The membrane was incubated with an appropriate mouse anti-His tag antibody for 2 hours and then washed three times with PBST buffer (12 minutes each). Protein detection was initiated by the addition of a pre-prepared developer.

[0071] (6) Determination of recombinant virus titer

[0072] The virus solution harvested after 10 generations of proliferation was cultured in DMEM medium containing 2% serum. -1 Dilute to 10 -10 . The diluted virus was inoculated into 96-well plates of cultured 293T cells. 100 μL of each dilution was inoculated and repeated in 8 wells. Observe the GFP changes and CPE of the virus in all wells every day and record the results for 2-4 days. The recorded data were used to calculate TCID 50 Results: The harvested viral seed stock was frozen and thawed 2-3 times, centrifuged to remove cell debris, and then centrifuged at 5000 rpm for 5 minutes at 4°C. The supernatant was then collected and the virus titer was determined. The virus with the highest titer was reserved as seed for batch production of live vector vaccines.

[0073] (7) Screening of the optimal cell line: The virus seed was inoculated into adherent culture cell lines (293T cell line, 293T cell line, 293LP cell line and AD293 cell line, etc.) and suspension culture cell lines (293F cell line and 293FP cell line, etc.), and the CPE of the virus was analyzed and its titer was determined. On this basis, the optimal conditions for virus reproduction were screened out, and the lowest-cost preparation method was calculated. Finally, 293FP suspension cells were selected for the culture of recombinant adenovirus. The optimal culture conditions were 37°C, 5% CO2 and 160 rpm / min.

[0074] (8) Preparation of recombinant live vector vaccine: According to the virus preparation method with the lowest cost and the best culture, recombinant virus was prepared in batches. The harvested virus liquid seed virus was repeatedly frozen and thawed three times, and then centrifuged to remove cell debris. The recombinant virus was then enriched by ultracentrifugation and resuspended in PBS to prepare the recombinant live vector vaccine for yellow croaker iridovirus disease.

[0075] Example 2:

[0076] In order to clarify the toxic and side effects of the recombinant live vector vaccine for preventing large yellow croaker iridovirus disease in the present invention when immunizing large yellow croaker, so as to make a scientific, accurate and objective evaluation of its safety, provide a basis for clinical immunization trials, and ensure clinical immunization safety, a safety test of the recombinant live vector vaccine for large yellow croaker iridovirus disease was conducted.

[0077] The recombinant live vector vaccine for large yellow croaker iridovirus disease used in this experiment was prepared using the method described in Example 1.

[0078] The safety test was conducted according to the Chinese Veterinary Pharmacopoeia. 90 yellow croakers were randomly divided into three groups, with 30 fish in each group (the weight of the fish was about 20 g). The first group of yellow croakers were soaked for 30 minutes and then diluted with 1×10 10.0 mL -1 TCID 50 The first group received a live vector vaccine. The second group received 0.1 mL of the live vector vaccine via intraperitoneal injection. The third group consisted of a control group of unvaccinated yellow croaker.

[0079] Clinical adverse reactions were observed in the three experimental groups, and fish deaths were collected daily for 14 days after immunization in the two experimental groups and the control group. The length and weight of fish in the two experimental groups and the control group were recorded for 60 days after immunization.

[0080] Within 14 days after vaccination, no fish died in either the immersion or injection immunization groups, or in the blank control group. Nor did any local or systemic adverse clinical reactions occur. Sixty days after vaccination, no significant differences in fish length or weight were observed in either immersion or injection immunization groups compared to the control group. This study demonstrates the safety of the recombinant live vector vaccine for yellow croaker iridovirus disease prepared by the present invention, providing experimental evidence for its application in the clinical immunization of yellow croaker.

[0081] Example 3:

[0082] In order to clarify the actual effect of the recombinant live vector vaccine for preventing large yellow croaker iridovirus disease in the present invention after immunizing large yellow croaker, so as to make a scientific and objective evaluation of its effectiveness and provide a basis for clinical immunization trials, an immune efficacy test of the recombinant live vector vaccine for large yellow croaker iridovirus disease was conducted.

[0083] The recombinant live vector vaccine for large yellow croaker iridovirus disease used in this experiment was prepared using the method described in Example 1.

[0084] (1) Inclusion criteria: Before the start of the immune efficacy test, the large yellow croaker was cultured under laboratory conditions for 10-14 days. No adverse clinical reactions were observed during the culture period. In addition, 10 fish were randomly selected for pathogen testing. No Pseudomonas aeruginosa, iridovirus, Cryptocaryon irritans, or other pathogens that may infect large yellow croaker were detected, which met the test requirements.

[0085] (2) Exclusion criteria: Fish that have been infected with a disease and have clinical symptoms are not included in this study. Secondly, if the pathogens are found in the fish during the etiology examination, such as Pseudomonas aeruginosa, iridovirus, and Cryptocaryon irritans, they are not included in this study.

[0086] (3) Effectiveness standard: After immunizing large yellow croaker with the recombinant live vector vaccine for large yellow croaker iridovirus disease of the present invention, there was no significant difference in the health status and growth status of the immunized large yellow croaker and the unvaccinated fish of the same age. No clinical adverse reactions occurred in the vaccinated large yellow croaker during the trial.

[0087] (4) Typical symptoms of large yellow croaker infected with iridovirus: In the early stage of infection, the body surface of the infected fish is intact, the fish swims slowly, and the food intake decreases. As the disease progresses, the gills of the infected fish become pale, the spleen and kidneys become enlarged, and the liver becomes congested. The fish refuses to eat until it dies.

[0088] Example 4:

[0089] Large yellow croaker immune efficacy test: 300 large yellow croakers were randomly divided into three groups, 100 in each group, with an average weight of about 20 g. The first group of fish was immunized with 1×10 10.0 mL -1 TCID 50 The live vector vaccine was diluted at a dilution of 1:100 and soaked for 30 min; the second group of fish was immunized with 1×10 10.0 mL -1 TCID 50 The empty vector control group virus was diluted at a dilution of 1:100 and soaked for 30 minutes; the third group was inoculated with DMEM medium as the control group.

[0090] On 15 dpv (days post vaccination), iridovirus was enriched and adjusted to a concentration of 100 TCID at 25 ± 1 °C. 50 (The TCID of the virus was determined 50 1×107.2 50 fish from each experimental group were randomly selected and immersed in the virus dilution solution for 30 minutes. Dead fish were collected daily for 30 days after iridovirus infection. Fish were observed for typical signs of iridovirus infection, such as sluggish swimming, decreased food intake, and pale gills. Necropsy revealed congestion in the liver, spleen, and kidneys. Dead fish were detected by PCR amplification of the ATPase gene (forward and reverse primers, see SEQ ID NO. 6 and SEQ ID NO. 7, respectively) to confirm that the deaths were due to iridovirus infection. The efficacy of the live vector vaccine was evaluated by comparing the total cumulative mortality (CPM) of fish across the three experimental groups. The relative percentage survival (RPS) value for each experimental group was calculated according to the following formula: RPS = [1 – (CPM of vaccinated fish / CPM of unvaccinated fish)].

[0091] The results are as follows Figure 4 As shown, the mortality rate of vaccinated fish was significantly lower than that of unvaccinated fish. The CPM of vaccinated large yellow croaker was 12.0%, while that of unvaccinated fish was 62.0%. Furthermore, the RPS (relative survival percentage) of vaccinated fish and the empty vector control group was 80.65% and 9.68%, respectively. Furthermore, the health and growth status of the vaccinated fish were not significantly different from those of normal fish of the same age. No adverse clinical reactions were observed in any of the fish during the trial.

[0092] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for preparing a recombinant live vector vaccine for large yellow croaker iridovirus disease, characterized in that: The following steps are involved: The MCP gene of large yellow croaker iridovirus was amplified and constructed into the pAd-Track-CMV shuttle vector. It was then recombined with the pAd-Easy-1 backbone vector in Escherichia coli to construct an MCP gene recombinant adenovirus plasmid. The recombinant adenovirus plasmid was linearized and transfected into the 293T cell line to obtain a replication-defective recombinant adenovirus. The positive recombinant adenovirus was inoculated into 293 suspension cells for multiple passages, and the stable high-titer recombinant adenovirus was selected to prepare a recombinant live vector vaccine for large yellow croaker iridovirus disease.

2. The method for preparing the recombinant live vector vaccine for large yellow croaker iridovirus disease according to claim 1, characterized in that: The nucleotide sequence of the MCP gene is shown in SEQ ID NO.1, and the nucleotide sequences of the primers used to amplify the MCP gene are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

3. The method for preparing the recombinant live vector vaccine for large yellow croaker iridovirus disease according to claim 1, characterized in that: The following steps are involved: (A) Amplification of the MCP gene The MCP gene was amplified from the genome of the large yellow croaker iridovirus. The upstream and downstream primers of the MCP gene were added with homologous sequences of the pAd-Track-CMV shuttle vector and Bgl II and Not I restriction sites, respectively. The nucleotide sequence of the MCP gene is shown in SEQ ID NO. 1, and the nucleotide sequences of the primers used to amplify the MCP gene are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively. (B) Rapid ligation of the MCP gene with the pAd-Track-CMV shuttle vector The cloned MCP gene was connected to the pAd-Track-CMV shuttle vector using rapid fusion technology to obtain a recombinant shuttle vector containing the MCP gene; (C) Homologous recombination between the pAd-Track-CMV shuttle vector and the pAd-Easy-1 backbone vector The pAd-Track-CMV shuttle vector carrying the MCP gene was recombined with the pAd-Easy-1 backbone vector in Escherichia coli to obtain a recombinant adenovirus plasmid named Adv-MCP. (D) Recombinant adenovirus packaging and preparation The recombinant plasmid Adv-MCP in step (C) was linearized by Pac Ⅰ digestion and then transfected into 293T cell line for virus packaging and preparation to obtain recombinant adenovirus; (E) Detection of recombinant virus antigen protein expression level Western-blot method was used to detect the expression of MCP protein to confirm that the constructed recombinant adenovirus could express the target protein; (F) Preparation of a recombinant live vector vaccine for yellow croaker iridovirus disease After multiple passages, the recombinant adenovirus that can express the target protein is harvested and the recombinant adenovirus with a stable and high virus titer is harvested. After repeated freezing and thawing, centrifugation, and re-proportioning, the recombinant live vector vaccine for yellow croaker iridovirus disease is prepared.

4. A recombinant live vector vaccine for large yellow croaker iridovirus disease, characterized in that: The vaccine is prepared by the method according to any one of claims 1 to 3.

5. Use of the recombinant live vector vaccine for large yellow croaker iridovirus disease as claimed in claim 4 in the preparation of an agent for preventing large yellow croaker iridovirus disease.

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