Pseudosciaena crocea iridovirus inactivated vaccine as well as preparation method and application thereof

Abstract

The invention provides a larimichthys crocea iridovirus inactivated vaccine as well as a preparation method and application thereof, and belongs to the technical field of preparation of viral vaccines. The larimichthys crocea iridovirus inactivated vaccine comprises an inactivated larimichthys crocea iridovirus and an adjuvant, and the strain of the larimichthys crocea iridovirus is an FD201807 strain. After the larimichthys crocea iridovirus inactivated vaccine is injected, the phenomena of ingestion and abnormal behaviors of fishes are avoided, the death phenomenon is avoided, the safety is high, the relative immune protection rate of the inactivated vaccine to immunized fishes is 51.20% or above, the immune effect is good, and the larimichthys crocea iridovirus inactivated vaccine can be applied to prevention and treatment of larimichthys crocea iridovirus diseases. The preparation method of the larimichthys crocea iridovirus inactivated vaccine is simple and convenient, easy to operate, low in cost and suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of virus vaccine preparation, and in particular relates to an inactivated vaccine of large yellow croaker iris virus and a preparation method and application thereof. Background Art

[0002] Large yellow croaker (Larimichthys crocea) is a marine fish with high aquaculture yield. Large yellow croaker iridovirus (LYCIV) belongs to the genus Megalocytivirus of the family Iridoviridae. At present, large yellow croaker iridovirus disease is difficult to prevent and control. The virus can cause explosive mortality of farmed large yellow croaker fry, causing serious economic losses to the large yellow croaker aquaculture industry. Large yellow croaker iridovirus and other iridoviruses of the genus Megalocytivirus have the same clinical symptoms in fish infected with diseased fish, such as slow reaction, abnormal swimming, enlarged spleen and kidney, etc. At present, the research on LYCIV mainly focuses on the establishment of virus detection methods, the analysis of some functional genes, and the cellular localization of some functional proteins. There are no reports on the research of virus vaccines and immune prevention and treatment. Summary of the invention

[0003] In view of this, the object of the present invention is to provide an inactivated vaccine of large yellow croaker iridovirus and a preparation method and application thereof, wherein the inactivated vaccine of large yellow croaker iridovirus has high safety and good immune effect.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides an inactivated vaccine of large yellow croaker iridovirus, comprising an inactivated large yellow croaker iridovirus and an adjuvant, wherein the strain of the large yellow croaker iridovirus is the FD201807 strain.

[0006] Preferably, the adjuvant is Montanide TM GEL 02PR adjuvant.

[0007] Preferably, the virus concentration of the large yellow croaker iridovirus is 1.0 to 8.0×10 8 copies / μL; the volume ratio of the large yellow croaker iridovirus to the adjuvant is 18:2-2.5.

[0008] Preferably, the method for preparing the inactivated large yellow croaker iridovirus comprises: mixing the FD201807 strain with β-propiolactone, inactivating the mixture, and treating the mixture in a water bath to obtain the inactivated large yellow croaker iridovirus.

[0009] Preferably, the inactivation method is inactivation at 2-6°C for 60-72h; and the water bath treatment method is water bath treatment at 36-40°C for 1.5-2.5h.

[0010] Preferably, the volume ratio of the FD201807 strain to β-propiolactone is 30 mL:30-45 μL.

[0011] The present invention provides a method for preparing the above-mentioned large yellow croaker iridovirus inactivated vaccine, comprising the following steps:

[0012] (1) The FD201807 strain was mixed with β-propiolactone, inactivated, and treated in a water bath to obtain an inactivated large yellow croaker iridovirus;

[0013] (2) The inactivated large yellow croaker iridovirus is mixed with an adjuvant to obtain an inactivated large yellow croaker iridovirus vaccine.

[0014] The present invention also provides an application of the FD201807 strain in the preparation of an inactivated vaccine of large yellow croaker iridovirus.

[0015] The present invention also provides a use of the above-mentioned large yellow croaker iridovirus inactivated vaccine in the preparation of products for preventing and treating diseases infected by large yellow croaker iridovirus and / or preventing and treating large yellow croaker iridovirus infection.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The invention provides an inactivated vaccine for yellow croaker iridovirus and a preparation method and application thereof. After injection of the inactivated vaccine for yellow croaker iridovirus, fish do not have feeding or abnormal behavior, and there is no death, and the safety is high. The relative immune protection rate of the inactivated vaccine for the immunized fish is above 51.20%, and the immune effect is good. The vaccine can be applied to the prevention and treatment of yellow croaker iridovirus disease. The preparation method of the inactivated vaccine for yellow croaker iridovirus is simple, easy to operate, low in cost, and suitable for industrial production. DETAILED DESCRIPTION

[0018] The invention provides an inactivated vaccine of large yellow croaker iridovirus, comprising an inactivated large yellow croaker iridovirus and an adjuvant, wherein the strain of the large yellow croaker iridovirus is the FD201807 strain.

[0019] In the present invention, the FD201807 strain was isolated from the kidney tissue of diseased juvenile large yellow croaker in Shacheng Sea Area, Fuding, Ningde, Fujian Province. It is a virus isolate belonging to the genus Megalocytivirus of the family Iridoviridae, and is named large yellow croaker iridovirus FD201807 in the present invention.

[0020] In the present invention, the adjuvant is preferably Montanide TM GEL 02PR adjuvant: The present invention has no special limitation on the source of MontanideTM GEL 02PR adjuvant, and any commercial product known in the art can be used.

[0021] In the present invention, the virus concentration of the large yellow croaker iridovirus is preferably 1.0 to 8.0×10 8 copies / μL, more preferably 1.5 to 4×10 8 copies / μL, more preferably 1.6×10 8 copies / μL, 2×10 8 copies / μL, 3×10 8 copies / μL or 4×10 8 The volume ratio of the large yellow croaker iridovirus to the adjuvant is preferably 18:2-2.5, and more preferably 18:2, 18:2.22 or 18:2.45. The large yellow croaker iridovirus inactivated vaccine of the present invention is a white liquid and is an injectable vaccine. After the inactivated vaccine is injected, the survival rate of farmed fish infected with the large yellow croaker iridovirus can be improved.

[0022] In the present invention, the preparation method of the inactivated large yellow croaker iridovirus comprises: mixing the FD201807 strain with β-propiolactone, inactivating it, and treating it in a water bath to obtain the inactivated large yellow croaker iridovirus. The inactivation method is preferably inactivating at 2-6°C for 60-72h, and more preferably inactivating at 4°C for 60h, 4°C for 65h, or 4°C for 72h. The water bath treatment method is 36-40°C water bath treatment for 1.5-2.5h, further 37-39°C water bath treatment for 1.5-2.5h, and more preferably 38°C water bath treatment for 2h. In the present invention, the volume ratio of the FD201807 strain to β-propiolactone is 30mL:30-45μL, more preferably 30mL:32-45μL, and more preferably 30mL:30μL, 30mL:39μL, or 30mL:45μL. The present invention utilizes the virus inactivator β-propiolactone to destroy the viral nucleic acid structure without directly acting on the protein, and the inactivated vaccine can maintain good immunogenicity. At the same time, the residual β-propiolactone can be hydrolyzed into non-toxic β-hydroxypropionic acid in a 36-40°C water bath, thereby ensuring that it is harmless to the body.

[0023] The present invention provides a method for preparing the above-mentioned large yellow croaker iridovirus inactivated vaccine, comprising the following steps:

[0024] (1) The FD201807 strain was mixed with β-propiolactone, inactivated, and treated in a water bath to obtain an inactivated large yellow croaker iridovirus;

[0025] (2) The inactivated large yellow croaker iridovirus is mixed with an adjuvant to obtain an inactivated large yellow croaker iridovirus vaccine.

[0026] The specific method involved in the above step (1) is consistent with the preparation method of inactivated large yellow croaker iridovirus, and will not be repeated here. In the above step (2), the present invention can use vortex mixing for 10 to 15 minutes for mixing.

[0027] The present invention utilizes the FD201807 strain as the active ingredient of the inactivated vaccine for large yellow croaker iridovirus. After injection of the inactivated vaccine, the fish do not have any adverse reactions and no deaths occur, and the vaccine has the advantage of high safety. Meanwhile, the inactivated vaccine can effectively prevent and treat the infection of large yellow croaker iridovirus or the diseases caused by the infection of fish (such as large yellow croaker or spotted sea bass).

[0028] Based on this, the present invention also provides an application of the above-mentioned FD201807 strain in the preparation of an inactivated vaccine of large yellow croaker iridovirus.

[0029] The present invention also provides a use of the above-mentioned large yellow croaker iridovirus inactivated vaccine in the preparation of products for preventing and treating diseases infected by large yellow croaker iridovirus and / or preventing and treating large yellow croaker iridovirus infection.

[0030] The product includes reagents or drugs. The administration method of the product is injection, such as intraperitoneal injection.

[0031] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] In the following examples, the large yellow croaker iridovirus was isolated from the kidney tissue of diseased large yellow croaker juveniles in Shacheng Sea Area, Fuding, Ningde, Fujian Province, and the FD201807 strain was obtained. The FD201807 strain belongs to the virus isolate of the genus Megalocytivirus of the family Iridoviridae and is named large yellow croaker iridovirus FD201807. The FD201807 strain has been publicly published in "Cell Culture and Comparison of Major Capsid Protein Genes of Different Strains of Large Yellow Croaker Megalocytivirus", Yang Xixi et al., Journal of Fisheries of China, 2023, 47(9):099412, DOI:10.11964 / jfc.20210813018.

[0034] Commercialization of Montanide by Seppic, France TMGEL 02PR adjuvant, hereinafter referred to as GEL 02PR.

[0035] Example 1

[0036] A method for preparing an inactivated vaccine of large yellow croaker iridovirus, comprising the following steps:

[0037] (1) Preparation of inactivated virus solution: Take 2.0×10 8 To 30 mL of the 100 copies / μL large yellow croaker iridovirus FD201807 virus solution, add 30 μL of β-propiolactone (the final volume concentration of β-propiolactone is 0.1%), mix well, let stand at 4°C for 72 h, and then place in a 38°C water bath for 2 h to obtain the inactivated virus solution.

[0038] (2) Take 18 mL of the inactivated virus solution, add 2 mL of GEL 02PR adjuvant, and vortex mix for 10 minutes to obtain the large yellow croaker iris virus inactivated vaccine.

[0039] The calculated virus load of the inactivated vaccine of yellow croaker iridovirus is 1.78×10 8 copies / μL.

[0040] Example 2

[0041] A method for preparing an inactivated vaccine of large yellow croaker iridovirus, comprising the following steps:

[0042] (1) Preparation of inactivated virus solution: Take 2.0×10 8 To 30 mL of the 100 copies / μL large yellow croaker iridovirus FD201807 virus solution, add 45 μL of β-propiolactone (the final volume concentration of β-propiolactone is 0.15%), mix well, let stand at 4°C for 65 h, and then place in a 38°C water bath for 2 h to obtain the inactivated virus solution.

[0043] (2) Take 18 mL of the inactivated virus solution, add 2.45 mL of GEL 02PR adjuvant, and vortex mix for 15 minutes to obtain the large yellow croaker iris virus inactivated vaccine.

[0044] Example 3

[0045] A method for preparing an inactivated vaccine of large yellow croaker iridovirus, comprising the following steps:

[0046] (1) Preparation of inactivated virus solution: Take 2.0×10 8 To 30 mL of the large yellow croaker iridovirus FD201807 virus solution (copies / μL), add 39 μL of β-propiolactone (the final volume concentration of β-propiolactone is 0.13%), mix well, let stand at 4°C for 60 h, and then place in a 38°C water bath for 2 h to obtain the inactivated virus solution.

[0047] (2) 18 mL of the inactivated virus solution was added to 2.22 mL of GEL 02PR adjuvant and vortexed for 13 min to obtain the large yellow croaker iris virus inactivated vaccine.

[0048] Example 4

[0049] (1) Detection of the inactivation effect of the inactivated virus solution of Example 1

[0050] 100 μL of inactivated virus solution was inoculated into the yellow croaker iridovirus-sensitive cell line, yellow croaker embryonic cell line type I (YCE1), and the cells were observed for 7 days without any lesions.

[0051] (2) Safety test of the inactivated vaccine of large yellow croaker iridovirus described in Example 1

[0052] A total of 120 large yellow croakers (weight 4-6 g) were evenly divided into 4 groups: a single-dose immunization group, a double-dose immunization group, a single-dose negative control group, and a double-dose negative control group. Each single-dose immunization group was intraperitoneally injected with 50 μL 1.78×10 8 Copies / μL Yellow croaker iridovirus inactivated vaccine, 100 μL 1.78×10 8 The single-dose negative control group and the double-dose negative control group were injected with 50 μL cell culture medium DMEM and 100 μL cell culture medium DMEM, respectively, and each group was injected once. The feeding and activity of the experimental fish were observed daily for 2 consecutive weeks.

[0053] The results showed that after injection of the inactivated yellow croaker iris virus vaccine, there was no abnormal eating or behavior in the yellow croaker, and no death, indicating that the inactivated yellow croaker iris virus vaccine is safe.

[0054] (3) Evaluation of the immune effect of inactivated vaccine against yellow croaker iridovirus

[0055] Evaluation of the immune effect on large yellow croaker: Large yellow croaker (weight 5-7 g) was obtained from a large yellow croaker nursery in Ningde, Fujian Province, and was not infected with iridescent virus. After temporary rearing, it was divided into two groups (control group and vaccine group), 100 fish in each group, and was reared in a white drum containing 600L seawater with a salinity of 20 and a water temperature of 20-22℃. Each fish in the control group was intraperitoneally injected with 50μL of cell culture medium, and each fish in the vaccine group was injected with 50μL of 1.78×10 8 copies / μL of the inactivated vaccine for the yellow croaker iridovirus described in Example 1. On the 21st day after immunization, 150 μL of the virus was injected intraperitoneally into each yellow croaker, with a volume of 2×10 8The virus solution of yellow croaker iridovirus FD201807 was used at 100 copies / μL for 2 consecutive weeks. The water temperature was maintained at 27-28℃ during the period. The mortality was observed and recorded, and the relative immune protection rate (RPS) was calculated according to the formula "RPS = (1-mortality rate of the immunized group / mortality rate of the control group) × 100%".

[0056] Evaluation of the immune effect on sea bass: Sea bass (weight 7-10 g) were obtained from a certain seedling technology company in Ningde, Fujian Province, and were not infected with iridescent virus. After temporary rearing, they were divided into two groups (control group and vaccine group), with 100 fish in each group, and were reared in a white drum containing 600L seawater with a salinity of 20 and a water temperature of 20-22℃. Each fish in the control group was intraperitoneally injected with 50μL of cell culture medium, and each fish in the vaccine group was injected with 50μL of 1.78×10 8 copies / μL of the inactivated vaccine for the yellow croaker iridovirus described in Example 1. On the 21st day after immunization, 50 μL of the virus was injected intraperitoneally into each yellow croaker, with a volume of 5.0×10 7 The virus liquid of yellow croaker iridovirus FD201807 was used at 100 copies / μL for 2 consecutive weeks. The water temperature was maintained at 22-24°C during the period. The mortality was observed and recorded, and the relative immune protection rate was calculated according to the formula "RPS = (1-mortality rate of the immune group / mortality rate of the control group) × 100%".

[0057] Table 1 Immune effect of inactivated vaccine against large yellow croaker iridovirus

[0058]

[0059] The results of immune effect evaluation in Table 1 show that the cumulative mortality of large yellow croaker and spotted seabass in the inactivated vaccine group was lower than that in the control group. The relative immune protection rates of large yellow croaker iridovirus inactivated vaccine against large yellow croaker and spotted seabass were 51.20% and 63.30%, respectively.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An inactivated vaccine for large yellow croaker iridovirus, characterized in that: The invention comprises inactivated large yellow croaker iridovirus and an adjuvant, wherein the strain of the large yellow croaker iridovirus is the FD201807 strain.

2. The inactivated vaccine of large yellow croaker iridovirus according to claim 1, characterized in that: The adjuvant is Montanide TM GEL 02PR adjuvant.

3. The inactivated vaccine of large yellow croaker iridovirus according to claim 1, characterized in that: The virus concentration of the large yellow croaker iridovirus is 1.0 to 8.0×10 8 copies / μL; the volume ratio of the large yellow croaker iridovirus to the adjuvant is 18:2-2.

5.

4. The inactivated vaccine of large yellow croaker iridovirus according to claim 1, characterized in that The preparation method of the inactivated large yellow croaker iridovirus comprises: mixing the FD201807 strain with beta-propiolactone, inactivating the mixture, and performing water bath treatment to obtain the inactivated large yellow croaker iridovirus.

5. The inactivated vaccine of large yellow croaker iridovirus according to claim 4, characterized in that: The inactivation method is to inactivate at 2-6°C for 60-72h; the water bath treatment method is to treat in a water bath at 36-40°C for 1.5-2.5h.

6. The inactivated vaccine of large yellow croaker iridovirus according to claim 4, characterized in that: The volume ratio of the FD201807 strain to β-propiolactone is 30mL:30-45μL.

7. A method for preparing the inactivated vaccine of large yellow croaker iridovirus according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) The FD201807 strain was mixed with β-propiolactone, inactivated, and treated in a water bath to obtain an inactivated large yellow croaker iridovirus; (2) The inactivated large yellow croaker iridovirus is mixed with an adjuvant to obtain an inactivated large yellow croaker iridovirus vaccine.

8. Use of the FD201807 strain described in claim 1 in preparing an inactivated vaccine of large yellow croaker iridovirus.

9. Use of the inactivated large yellow croaker iridovirus vaccine according to any one of claims 1 to 6 in the preparation of products for preventing and treating diseases caused by large yellow croaker iridovirus infection and / or preventing and treating large yellow croaker iridovirus infection.

Images