Aquatic animal vaccine transgenic organism safety evaluation method

Abstract

The invention relates to an aquatic animal vaccine safety evaluation method, in particular to an aquatic animal vaccine transgenic organism safety evaluation method. The method comprises the following steps: 1, immunizing cold water mode fish; and 2, nucleic acid vaccine environment retention and transfer detection. According to the safety evaluation method disclosed by the invention, by analyzing the recombinant plasmid content of rainbow trout excrement and a water body environment after immunizing nucleic acid vaccine plasmids, the condition of transferring plasmids to the environment outside an immune animal individual is explored. Whether bacteria in the environment where the immunized rainbow trout is located can obtain antibiotic resistance from the plasmid pIHNch-G or not is detected, an experiment is conducted with escherichia coli as indicator bacteria, and then safety evaluation of aquatic animal vaccine transgenic organisms is achieved. The method is easy to operate, high in safety and convenient to popularize and use.

Description

Technical Field

[0001] The invention relates to a method for evaluating the safety of aquatic animal vaccines. Background Art

[0002] Vaccines are an effective means of fighting against diseases. As a new type of vaccine, genetically engineered vaccines have the advantages of safety, high efficiency, low cost, and easy creation of multi-component and multivalent vaccines. In recent years, they have gradually become the main type of vaccine research and development. Unlike traditional inactivated vaccines, genetically engineered vaccines require genetically modified organism safety assessments. Aquatic animals live in water, and compared with terrestrial animals, the research sites and technologies for genetically modified organism safety assessments are special. Aquatic genetically modified organisms are strictly prohibited from entering the environment before obtaining a genetically modified organism safety certificate, so the requirements for genetically modified organism safety assessment sites and breeding facilities are relatively high. Summary of the invention

[0003] The invention provides a method for evaluating the safety of transgenic organisms in aquatic animal vaccines.

[0004] The method for evaluating the safety of transgenic organisms in aquatic animal vaccines of the present invention is carried out according to the following steps:

[0005] 1. Immunization of cold-water model fish: The cold-water model fish were randomly divided into two groups: a blank group and an immunized group. The blank control group was injected with 0.05 ml / tail of PBS buffer solution, and the immunized group was injected with the nucleic acid vaccine to be tested into the muscle at the base of the dorsal fin. The two groups of cold-water model fish were then placed in a water tank for breeding. The dose of the nucleic acid vaccine to be tested was 0.05 ml / tail.

[0006] 2. Nucleic acid vaccine environmental retention and transfer detection: Add 200 mL of bacterial cells with a concentration of 1×10 7 cfu / mL of Escherichia coli DH5α suspension, take water samples and bottom feces at different growth days, extract DNA, and then use PCR method to detect the presence of nucleic acid vaccine plasmid DNA in rainbow trout breeding water environment and feces, and monitor whether the feces and water environment of immune rainbow trout contain recombinant plasmids of nucleic acid vaccine;

[0007] If no nucleic acid vaccine plasmid is detected, the nucleic acid vaccine has not been transferred to the external environment of the immunized animal through the feces; if the nucleic acid vaccine plasmid is detected, the nucleic acid vaccine has been transferred to the external environment of the immunized animal through the feces; thereby determining the safety of the genetically modified organism of the aquatic animal vaccine to be tested.

[0008] Further testing: The water environment and feces of the cold water model fish farming are screened with resistance plates, and samples of colonies that can grow on the resistance plates are selected for 16S PCR sequencing and identification, and plasmids are extracted from the resistant bacteria identified as Escherichia coli, and then the plasmids are sequenced to determine whether the plasmids contain the nucleic acid vaccine gene sequence; thereby further determining the safety of the genetically modified organisms of the aquatic animal vaccine to be tested; the resistance plates contain antibiotics corresponding to the nucleic acid vaccine.

[0009] The safety evaluation method of the present invention analyzes the recombinant plasmid content in the feces and water environment of rainbow trout after immunization with nucleic acid vaccine plasmids, and explores the transfer of plasmids to the environment outside the immunized animal. It is tested whether bacteria in the environment of rainbow trout after immunization can obtain antibiotic resistance from the plasmid pIHNch-G, and experiments are carried out with Escherichia coli as indicator bacteria, thereby realizing the safety evaluation of transgenic organisms of aquatic animal vaccines. The method of the present invention is simple to operate and has high safety, so it is easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The PCR results of DNA extracted from water samples and intestinal contents 1 day after vaccine immunization in Example 1;

[0011] Figure 2 The PCR results of DNA extracted from water samples, feces, and intestinal contents 3 days after vaccine immunization in Example 1;

[0012] Figure 3 The PCR results of DNA extracted from water samples, feces, and intestinal contents 5 days after vaccine immunization in Example 1;

[0013] Figure 4 The PCR results of DNA extracted from water samples, feces, and intestinal contents 7 days after vaccine immunization in Example 1;

[0014] Figure 5 The PCR results of DNA extracted from water samples, feces, and intestinal contents 14 days after vaccine immunization in Example 1;

[0015] Figure 6 The PCR results of DNA extracted from water samples, feces, and intestinal contents 21 days after vaccine immunization in Example 1;

[0016] Figure 7 The PCR results of DNA extracted from water samples, feces, and intestinal contents 28 days after vaccine immunization in Example 1;

[0017] Figure 8 The PCR results of DNA extracted from water samples, feces, and intestinal contents 35 days after vaccine immunization in Example 1;

[0018] Fig. 9The PCR results of DNA extracted from water samples, feces, and intestinal contents 42 days after vaccine immunization in Example 1;

[0019] Fig.10 The PCR results of DNA extracted from water samples, feces, and intestinal contents 49 days after vaccine immunization in Example 1;

[0020] Fig.11 The PCR results of DNA extracted from water samples, feces, and intestinal contents 56 days after vaccine immunization in Example 1;

[0021] Fig.12 These are the PCR results after DNA was extracted from water samples, feces, and intestinal contents 65 days after vaccine immunization in Example 1. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments 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 creative work are within the scope of protection of the present invention.

[0023] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0024] Specific implementation method 1: The method for evaluating the safety of transgenic organisms in aquatic animal vaccines in this implementation method is carried out according to the following steps:

[0025] 1. Immunization of cold-water model fish: The cold-water model fish were randomly divided into two groups: a blank group and an immunized group. The blank control group was injected with 0.05 ml / tail of PBS buffer solution, and the immunized group was injected with the nucleic acid vaccine to be tested in the muscle at the base of the dorsal fin. The two groups of cold-water model fish were then placed in a water tank for breeding. The dose of the nucleic acid vaccine to be tested was 0.05 ml / tail (2 μg / tail).

[0026] 2. Nucleic acid vaccine environmental retention and transfer detection: Add 200 mL of bacterial cells with a concentration of 1×10 7 cfu / mL of Escherichia coli DH5α suspension, take water samples and bottom feces at different growth days, extract DNA, and then use PCR method to detect the presence of nucleic acid vaccine plasmid DNA in rainbow trout breeding water environment and feces, and monitor whether the feces and water environment of immune rainbow trout contain recombinant plasmids of nucleic acid vaccine;

[0027] If no nucleic acid vaccine plasmid is detected, the nucleic acid vaccine has not been transferred to the external environment of the immunized animal through the feces; if the nucleic acid vaccine plasmid is detected, the nucleic acid vaccine has been transferred to the external environment of the immunized animal through the feces; thereby determining the safety of the genetically modified organism of the aquatic animal vaccine to be tested.

[0028] In step 1 of this embodiment, the 0.05 ml nucleic acid vaccine dose injected into each tail contains 2 μg of nucleic acid vaccine.

[0029] In step 2 of this embodiment, the different growth day time points are 1, 3, 5, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 65 days.

[0030] Specific implementation method 2: This implementation method is different from specific implementation method 1 in that: further testing: taking cold water model fish breeding water environment and feces for screening with resistance plates, selecting colonies that can grow on the resistance plates for sampling and 16S PCR sequencing identification, and extracting plasmids from resistant bacteria identified as Escherichia coli, and then sequencing the plasmids to determine whether the plasmids contain nucleic acid vaccine gene sequences; thereby further determining the safety of the transgenic organisms of the aquatic animal vaccine to be tested; the resistance plates contain antibiotics corresponding to the nucleic acid vaccine. Other steps and parameters are the same as those of specific implementation method 1.

[0031] In this embodiment, the plate coated with resistance is an ampicillin-resistant and / or kanamycin-resistant plate.

[0032] Specific implementation method three: This implementation method is different from the specific implementation method one in that the cold water model fish in step one is rainbow trout. The other steps and parameters are the same as those in the specific implementation method one.

[0033] Specific implementation method 4: This implementation method is different from specific implementation method 1 in that: in step 1, the cold water mode fish rearing environment: select a separate pond or a drained pond, do not raise any fish in the adjacent pond, and have fences and protective nets around to effectively prevent birds from entering and exiting at will; formulate an entry and exit management system, set door locks, and manage by a dedicated person; configure monitoring equipment to monitor the pond status in real time 24 hours a day without blind spots. Other steps and parameters are the same as those in specific implementation method 1.

[0034] Specific implementation method 5: This implementation method is different from specific implementation method 1 in that: the two groups of cold water model fish described in step 1 are placed in a water tank for breeding, that is, in a stainless steel circulating water tank, with running water outside the water tank for cooling, the water tank is isolated from the running water, and there is no water exchange; after the circulating water in the water tank is circulated, the discharged wastewater is disinfected in the secondary treatment tank and discharged after passing the test; and there are two groups of secondary treatment tanks, which can meet the disinfection treatment requirements of the discharged water. Other steps and parameters are the same as those in specific implementation method 1.

[0035] Specific embodiment 6: This embodiment differs from specific embodiment 1 in that: in step 2, the PCR method uses primer pairs for antigen protein G gene IHNV-G / F and IHNV-G / R, kanamycin resistance gene Neo RF / Neo R r, and ampicillin resistance gene AMPr 1-F / AMPr 1-R;

[0036] The primer sequence of antigen protein G gene is

[0037] 5'-GGATCCCTTTTGTGCTTTGAGACCGAACGCA-3',

[0038] 5'-CTCGAGGTGCCTCCATGTTCGTGTTTATGTC-3'; the kanamycin resistance gene primer sequence is 5'-ATGATTGAACAAGATGGATTGCACG-3',

[0039] 5'-TCAGAAGAACTCGTCAAGAAGGCGA-3'; the primer sequence of ampicillin resistance gene is 5'-TTACCAATGCTTAATCAGTGAGGCA-3',

[0040] 5'-ATGAGTATTCAACATTTCCGTGTCG-3'. Other steps and parameters are the same as those in the first embodiment.

[0041] Example 1 Safety evaluation of rainbow trout from Heilongjiang using the method of the present invention

[0042] The rainbow trout used in this embodiment are selected to be cultured in separate ponds or drainage ponds. No fish are raised in adjacent ponds, and there are fences and protective nets around them to effectively prevent birds and other animals from entering and leaving at will; formulate an entry and exit management system, set door locks, and manage them by dedicated personnel; configure monitoring equipment to monitor the status of the pond in real time 24 hours a day without dead ends. The two groups of test fish, the blank group and the immune group, are cultured in a stainless steel circulating water tank. There is running water outside the water tank for cooling. The water tank is isolated from the running water and there is no water exchange. After the circulating water in the water tank is circulated, the discharged wastewater is disinfected in the secondary treatment tank and discharged after passing the test. And there are two groups of secondary treatment tanks, which can effectively respond to the disinfection treatment needs of the discharged water body.

[0043] Specific methods:

[0044] 1. Immunization of the Animals to be Tested 5±1g rainbow trout were temporarily kept in a water tank (90cm in diameter and 60cm in height), fasted for 2 days before the test, and then injected with nucleic acid vaccine at the base of the dorsal fin. During the immunization, the rainbow trout were randomly divided into two groups: a blank group and an immunized group, with 1000 trout in each group. The immunization dose of the nucleic acid vaccine was 0.05ml / tail (the content of plasmid pIHNch-G was 2μg); the blank control group was injected with 0.05ml / tail PBS buffer solution. After immunization, the rainbow trout were placed in a circulating water tank in an environmental release pond for breeding, as shown in Table 1.

[0045] Table 1 Grouping of rainbow trout nucleic acid vaccine environmental retention test after immunization

[0046]

[0047] 2. Detection of environmental retention and transfer of nucleic acid vaccines

[0048] After the immunization, the rainbow trout in each group were cultured in a circulating water tank, and 1×10 7 cfu / mL or above, and create an artificial favorable transfer environment. Water samples and bottom feces residues were collected at various time points (1, 3, 5, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 65 days) to extract DNA. The presence of nucleic acid vaccine plasmid DNA in rainbow trout aquaculture water environment and feces was detected by PCR using primer pairs IHNV-G / F, IHNV-G / R, AMPr 1-F / AMPr 1-R, and Neo RF / Neo R r. The presence of recombinant plasmid pIHNch-G in the feces and water environment of immune rainbow trout was monitored to explore the transfer of plasmids to the environment outside the immune animal through feces and other pathways. The primer sequences are shown in Table 2.

[0049] Table 2 Primer sequences

[0050]

[0051]

[0052] 3. Detection of transfer of nucleic acid vaccine plasmid resistance genes to the environment

[0053] The DNA extracted in step 2 is transformed into Escherichia coli DH5ɑ, and then coated on a resistance plate and cultured at 37°C. If the results show that no colonies grow on all plates, while the positive control is full of resistant colonies, it is determined that the resistance gene comes from the environment rather than the nucleic acid vaccine to be tested, that is, it is determined that the cold water model fish is immunized with the vaccine to be tested, and no transfer of the nucleic acid vaccine to the environment is found; the water samples and feces to be obtained are screened with ampicillin resistance plates to see whether the Escherichia coli remaining in the environment can grow on the resistance plates; the colonies grown on the resistance plates are sampled for 16S PCR sequencing identification, and the resistant bacteria identified as Escherichia coli are subjected to plasmid extraction. If a plasmid is obtained, the plasmid is sequenced to determine whether the plasmid resistance gene in the immune rainbow trout can be transferred to the non-resistant Escherichia coli in the environment.

[0054] That is, the PCR method is used to identify whether the recombinant plasmid pIHNch-G exists in the sample, and the target genes are the glycoprotein gene (G) carried by the recombinant plasmid, as well as the ampicillin resistance gene (AMP) and the kanamycin resistance gene (Neo).

[0055] The materials used in this example are as follows:

[0056] Nucleic acid vaccine plasmid and strain immunization Nucleic acid vaccine plasmid was prepared and provided by Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences (Xu L, Zhao J, Liu M, et al. A effective DNA vaccine against diverse genotype Jinfectious hematopoietic necrosis virus strains prevalent in China. Vaccine, 2017, 35(18): 2420-2426). Escherichia coli DH5α was purchased from Bao Biotechnology (Dalian) Co., Ltd.

[0057] Self-prepared reagents: Solution I for vaccine purification: glucose 9.9 g / l, Tris-Cl 3.025 g / l, EDTA 3.72 g / l, RNase A 0.2 g / l. Solution II for vaccine purification: 1% SDS 320 ml, 10 M NaOH 6.4 ml. Solution III for vaccine purification: potassium acetate 294 g / l, glacial acetic acid 115 ml / l. Feed for fermentation: tryptone 30 g / l, yeast extract 15 g / l, glycerol 50% (v / v). All reagents used were purchased from Aladdin Reagent Co., Ltd.

[0058] Culture medium and reagents: Glycerol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd., Tryptone and yeast extract in the LB liquid culture medium formula were purchased from OXOID Company, and sodium chloride was purchased from Aladdin Reagent Co., Ltd.

[0059] Experimental instruments include clean bench, 3K15 high-speed refrigerated centrifuge (Sigma), water bath, constant temperature shaker, constant temperature incubator, etc.

[0060] Results of this example:

[0061] 1. Retention of recombinant plasmid pIHNch-G in aquaculture water and rainbow trout feces

[0062] According to the above-mentioned time points, the aquaculture water and feces at the bottom of the pond were collected (no feces 1 day after immunization), DNA was extracted, and the PCR method was used to identify whether the recombinant plasmid pIHNch-G existed in the sample. The target genes were the glycoprotein gene (G) and the ampicillin resistance gene (AMP) and kanamycin resistance gene (Neo) carried by the recombinant plasmid. The recombinant plasmid was used as the positive control, and the source water of the aquaculture pond was used as the blank control. The results of gel electrophoresis analysis of the PCR amplification products showed that specific target bands appeared in the positive control plasmids at each time point, including 1694bp for the G gene, 861bp for the AMP resistance gene, and 795bp for the Neo resistance gene; the blank control did not have any DNA bands, indicating that the detection was established. The gel electrophoresis detection results of this example are as follows: Figures 1 to 12 As shown, Figure 1 The PCR results of DNA extracted from water samples and intestinal contents 1 day after vaccine immunization in this example; Figure 2 The PCR results of DNA extracted from water samples, feces, and intestinal contents 3 days after vaccination in this example; Figure 3 The PCR results of DNA extracted from water samples, feces, and intestinal contents 5 days after vaccination in this example; Figure 4 The PCR results of DNA extracted from water samples, feces, and intestinal contents 7 days after vaccination in this example; Figure 5 The PCR results of DNA extracted from water samples, feces, and intestinal contents 14 days after vaccination in this example; Figure 6 The PCR results of DNA extracted from water samples, feces, and intestinal contents 21 days after vaccination in this example; Figure 7 The PCR results of DNA extracted from water samples, feces, and intestinal contents 28 days after vaccination in this example; Figure 8 The PCR results of DNA extracted from water samples, feces, and intestinal contents 35 days after vaccination in this example; Fig. 9 The PCR results of DNA extracted from water samples, feces, and intestinal contents 42 days after vaccination in this example; Fig.10The PCR results of DNA extracted from water samples, feces, and intestinal contents 49 days after vaccination in this example; Fig.11 The PCR results of DNA extracted from water samples, feces, and intestinal contents 56 days after vaccination in this example; Fig.12 The PCR results of DNA extracted from water samples, feces, and intestinal contents 65 days after vaccination in this example; Figure 1 M: DL2000 marker; 1: negative control; 2: positive control; 3, 4: water samples and intestinal contents of the experimental group; 5, 6: water samples and intestinal contents of the control group; Figures 2 to 12 In the figure: DL2000 marker; 1: negative control; 2: positive control; 3, 4, 5: water samples, feces, and intestinal contents of the experimental group; 6, 7, 8: water samples, feces, and intestinal contents of the control group.

[0063] The results of gel electrophoresis showed that no G gene was detected in water samples, feces, and intestinal contents at each time point ( Figures 1 to 12 ), but except for 1d and 3d after immunization, AMP resistance gene and / or Neo resistance gene fragments were detected in samples of each group at different time points ( Figures 3 to 12 ). Since the nucleic acid vaccine antigen protein G gene was not detected, it can be inferred that the detected resistance gene fragment did not come from the nucleic acid vaccine. To further prove this speculation, the present embodiment extracted the resistant microbial plasmid to transform Escherichia coli DH5ɑ, coated the corresponding resistance culture plate, and cultured it at 37°C. The results showed that no colonies grew on all plates, while the Escherichia coli DH5ɑ plate transformed with the positive control extracted plasmid was full of resistant colonies.

[0064] Since the transformation is carried out using a plasmid extract of a resistant microorganism, if the bacteria is an acquired nucleic acid vaccine gene, the nucleic acid vaccine plasmid will be extracted when the plasmid is extracted and transformed into Escherichia coli DH5ɑ, showing resistance, and colonies will appear in the resistance culture plate. However, since natural wild resistant bacteria may not have natural plasmids, and resistance genes will not exist in plasmids, Escherichia coli DH5ɑ transformed by plasmid extracts does not have resistance. The results of this embodiment further prove that the resistance genes in the sample come from the environment rather than the nucleic acid vaccine. In summary, the method of the present invention has determined that rainbow trout (5±1g) was immunized with this immunization protocol, and no transfer of nucleic acid vaccines to the environment was found.

[0065] 2. Transfer of the recombinant plasmid pIHNch-G resistance gene to environmental microorganisms

[0066] At the above time points, the aquaculture water, feces at the bottom of the pond (no feces 1 day after immunization) and intestinal contents were collected, and after centrifugation, the suspension was prepared and coated with ampicillin-resistant and kanamycin-resistant plates, and inverted at 37°C and 20°C, respectively. The colony formation was observed every day and the number of colonies was recorded. The results showed that because E. coli is a non-resistant bacterium, the lack of E. coli on the resistant plate indicated that the nucleic acid vaccine was not transferred to E. coli. No E. coli with ampicillin resistance or kanamycin resistance was isolated from the samples of immunized rainbow trout and control rainbow trout, but 60 other kanamycin-resistant strains and 30 ampicillin-resistant strains were isolated. Kanamycin-resistant strains included: 33 strains of Staphylococcus epidermidis strain Fussel (GenBank: NR_036904.1), 12 strains of Staphylococcus haemolyticus strain JCM 2416 (GenBank: NR_113345.1), and 15 strains of Staphylococcus capitis strain JCM 2420 (GenBank: NR_113348.1); ampicillin-resistant strains included: 20 strains of Pseudomonas (Pseudomonas koreensis strain Ps9-14 (GenBank: KU041145.1)), 4 strains of Aeromonas allosaccharophila strain K1 (GenBank: MK548513.1), and 2 strains of Enterobacter ludwigii strain GG33) (GenBank: KJ850208.1), Aeromonas salmonicida strain CECT 894 (GenBank: NR_043324.1). This result shows that the nucleic acid vaccine resistance gene has not been transferred to environmental microorganisms.

Claims

1. A method for evaluating the safety of transgenic organisms in aquatic animal vaccines, characterized in that The safety assessment method for genetically modified organisms in aquatic animal vaccines is carried out in the following steps:

1. Immunization of cold-water model fish: The cold-water model fish were randomly divided into two groups: a blank group and an immunized group. The blank control group was injected with 0.05 ml / tail of PBS buffer solution, and the immunized group was injected with the nucleic acid vaccine to be tested into the muscle at the base of the dorsal fin. The two groups of cold-water model fish were then placed in a water tank for breeding. The dose of the nucleic acid vaccine to be tested was 0.05 ml / tail.

2. Nucleic acid vaccine environmental retention and transfer detection: Add 200 mL of bacterial cells with a concentration of 1×10 7 cfu / mL of Escherichia coli DH5α suspension, take water samples and bottom feces at different growth days, extract DNA, and then use PCR method to detect the presence of nucleic acid vaccine plasmid DNA in rainbow trout breeding water environment and feces, and monitor whether the feces and water environment of immune rainbow trout contain recombinant plasmids of nucleic acid vaccine; If no nucleic acid vaccine plasmid is detected, the nucleic acid vaccine has not been transferred to the external environment of the immunized animal through the feces; if the nucleic acid vaccine plasmid is detected, the nucleic acid vaccine has been transferred to the external environment of the immunized animal through the feces; thereby determining the safety of the genetically modified organism of the aquatic animal vaccine to be tested.

2. The method for evaluating the safety of transgenic organisms in aquatic animal vaccines according to claim 1, characterized in that: Further testing: The water environment and feces of the cold water model fish farming are screened with resistance plates, and samples of colonies that can grow on the resistance plates are selected for 16S PCR sequencing and identification, and plasmids are extracted from the resistant bacteria identified as Escherichia coli, and then the plasmids are sequenced to determine whether the plasmids contain the nucleic acid vaccine gene sequence; thereby further determining the safety of the genetically modified organisms of the aquatic animal vaccine to be tested; the resistance plates contain antibiotics corresponding to the nucleic acid vaccine.

3. A method for evaluating the safety of transgenic organisms in aquatic animal vaccines according to claim 1, characterized in that The cold water model fish in step one is rainbow trout.

4. A method for evaluating the safety of transgenic organisms in aquatic animal vaccines according to claim 1, characterized in that Step 1: Cold water fish rearing environment: select a separate pond or a drained pond. Do not raise any fish in adjacent ponds and have fences and protective nets around them to effectively prevent birds from entering and exiting at will. Develop an entry and exit management system, set door locks, and assign dedicated personnel to manage the environment. Configure monitoring equipment to monitor the status of the pond in real time 24 hours a day without blind spots.

5. The method for evaluating the safety of transgenic organisms in aquatic animal vaccines according to claim 1, characterized in that The two groups of cold water model fish described in step one are raised in a water tank, that is, in a stainless steel circulating water tank, with running water outside the water tank for cooling, the water tank is isolated from the running water, and there is no water exchange; after the circulating water in the water tank is circulated, the discharged wastewater is disinfected in a secondary treatment tank and discharged after passing the test; and there are two groups of secondary treatment tanks, which can meet the disinfection treatment needs of the discharged water.

6. A method for evaluating the safety of transgenic organisms in aquatic animal vaccines according to claim 1, characterized in that In the PCR method in step 2, primer pairs are used for antigen protein G gene IHNV-G / F and IHNV-G / R, kanamycin resistance gene NeoR F / Neo R r, and ampicillin resistance gene AMPr 1-F / AMPr 1-R; The primer sequence of antigen protein G gene is 5'-GGATCCCTTTTGTGCTTTGAGACCGAACGCA-3', 5'-CTCGAGGTGCCTCCATGTTCGTGTTTATGTC-3'; the kanamycin resistance gene primer sequence is 5'-ATGATTGAACAAGATGGATTGCACG-3', 5'-TCAGAAGAACTCGTCAAGAAGGCGA-3'; the primer sequences of ampicillin resistance gene are 5'-TTACCAATGCTTAATCAGTGAGGCA-3', 5'-ATGAGTATTCAACATTTCCGTGTCG-3'.

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