Preparation method and preparation device of turbot edwardsiella tarda inactivated vaccine

Abstract

The invention relates to the technical field of bioengineering, and discloses a preparation method and a preparation device of a turbot edwardsiella tarda inactivated vaccine, the preparation device comprises a bottom plate, a storage assembly is fixedly mounted at the top of the bottom plate, a constant-temperature culture assembly is fixedly mounted at the top of the bottom plate, and a detection assembly is fixedly mounted at the top of the constant-temperature culture assembly. A uniform mixing assembly is fixedly mounted in the constant-temperature culture assembly. According to the preparation method and the preparation device of the turbot edwardsiella tarda inactivated vaccine, when the bacterial count is 15.0-60.0 * 10 < 8 > CFU / mL, 0.05% (V / V) of formalin can be added for inactivation (generally, the addition amount is not lower than 0.5%), and stimulation of formalin to fish bodies in the later period is reduced; if the addition amount of formalin is 0.5%, after inactivation, the formalin needs to be washed with sterile normal saline for multiple times and centrifuged, and the workload is increased and the possibility of pollution of other bacteria is easily increased in the process. And the overall immune challenge protection rate can be improved, and the immune effect is higher than that of a conventional antigen.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a method for preparing an Edwardsiella tarda inactivated vaccine of turbot and a preparation device thereof. Background Art

[0002] Turbot is a farmed species with high economic value. It grows quickly and tastes delicious, making it a popular species. Since it was introduced to China from Europe in the 1990s, turbot farming has developed rapidly. However, with the continuous deterioration of the farming environment, bacterial diseases of turbot continue to occur, and Edwardsiella disease is a common disease.

[0003] Edwardsiella disease is caused by Edwardsiella tarda, also known as Edwardsiella tarda or Edwardsiella slow, which belongs to the Enterobacteriaceae family. It is an extremely harmful pathogen in my country's aquaculture, causing huge economic losses to farmers and seriously affecting the healthy development of the turbot industry. At present, the main prevention and control method for bacterial diseases of turbot is the use of disinfectants and antibiotics, but these two methods have hidden dangers such as drug residues and bacterial resistance. In early 2019, the Ministry of Agriculture and Rural Affairs and ten other ministries jointly issued a programmatic document entitled "Several Opinions on Accelerating the Green Development of Aquaculture", emphasizing that in the prevention and control of aquaculture diseases, prevention should be the main focus, prevention and treatment should be combined, and preventive technologies such as vaccination should be promoted to promote green development of aquaculture.

[0004] Therefore, it is urgent to develop an effective, low-irritation, and residue-free vaccine for Edwardsiella tarda disease in turbot. Inactivated vaccine is a relatively safe and effective method for the prevention and control of aquatic diseases. The principle is to inactivate microorganisms through physical or chemical methods while maintaining the immunogenicity of the microorganism. After vaccination, the animal body can develop specific immunity to the microorganism.

[0005] In the prior art, although the bacteria can be inactivated, it is necessary to add not less than 0.5% formalin during the inactivation process, which may easily cause formalin to irritate the fish body in the later stage. In addition, the amount of formalin added is 0.5%. After inactivation, it is necessary to wash and centrifuge the fish several times with sterile saline, which increases the workload and the possibility of contamination by other bacteria. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a preparation method and a preparation device of an Edwardsiella tarda inactivated vaccine for turbot, which can effectively solve the problems in the prior art that during the inactivation process, not less than 0.5% of formalin needs to be added, which easily causes formalin to irritate the fish body in the later stage, and the amount of formalin added is 0.5%, and after inactivation, it is necessary to wash with sterile physiological saline for many times and centrifuge, and the workload is increased and the possibility of contamination by other bacteria is easily increased.

[0007] The technical solution adopted by the present invention is: a preparation device for an inactivated Edwardsiella tarda vaccine of turbot, comprising a bottom plate, a storage component is fixedly installed on the top of the bottom plate, a constant temperature culture component is fixedly installed on the top of the bottom plate, a detection component is fixedly installed on the top of the constant temperature culture component, a mixing component is fixedly installed inside the constant temperature culture component, a constant temperature storage box body is fixedly installed on the top of the bottom plate, a stirring component is fixedly installed inside the constant temperature storage box body, and a detection component is fixedly installed on the top of the bottom plate;

[0008] The storage assembly comprises a refrigerator body, a storage bar is plugged and installed inside the refrigerator body, a through slot is penetrated through the top of the storage bar, and a culture medium body is arranged on the top of the storage bar.

[0009] Preferably, the constant temperature incubation component includes a constant temperature incubator body, a display screen is provided on the front side of the constant temperature incubator body, a control switch is provided on the front side of the constant temperature incubator body, a partition bar is fixedly installed inside the constant temperature incubator body, a storage groove is provided at the bottom of the constant temperature incubator body, a card slot 1 is provided at the bottom of the constant temperature incubator body, a mounting plate is provided inside the storage groove, a fixing bolt 1 is fixedly installed on the top of the mounting plate, the constant temperature incubator body is plug-connected with the mounting plate through the storage groove and the card slot 1, and the fixing bolt 1 penetrates the mounting plate and extends to the interior of the constant temperature incubator body.

[0010] Through the above technical scheme, through the cooperation between the storage slot and the card slot 1 and the mounting plate, the mounting plate can be quickly aligned during installation, and then the fixing bolt 1 can be rotated to achieve its rapid installation, which can facilitate the later maintenance and inspection of the servo motor 1.

[0011] Preferably, the detection component includes a mounting seat, a mounting groove is provided on the inner side of the mounting seat, a slot one is provided on the inner side of the mounting seat, a sealing gasket is fixedly installed on the top of the mounting seat, a fixing bolt two is arranged on the top of the mounting seat, a connecting column one is plugged and installed inside the slot one, a probe is fixedly installed on the bottom of the connecting column one, a temperature detector body is fixedly installed on the top of the connecting column one, the probe passes through the mounting seat and extends to the interior of the constant temperature incubator body, and the detection component is located on the top of the constant temperature incubator body.

[0012] Through the above technical solution and the design of the detection component, when the bacterial solution is cultured at a constant temperature, the probe can be inserted into the interior of the constant temperature incubator body to detect the internal temperature, and then the detected data is transmitted to the surface of the temperature detector body for display, so that the staff can understand the production temperature of the bacterial solution.

[0013] Preferably, the mounting seat is matched with the connecting column 1 through the slot 1, the sealing gasket is a rubber sealing gasket, and the fixing bolt 2 passes through the mounting seat and extends to the interior of the constant temperature incubator body.

[0014] Through the above technical solution, through the cooperation between the mounting seat, slot one and connecting column one, when installing the component above connecting column one, connecting column one can be inserted through slot one, which can play a role of rapid alignment, and combined with the sealing gasket, it can prevent bacteria from entering the interior, causing the sterile environment of the bacteria to be affected, affecting the normal growth of the bacteria.

[0015] Preferably, the mixing component includes a protective cover, a protective cover is installed on the top of the protective cover, a servo motor 1 is fixedly installed inside the constant temperature incubator body, a fixed plate is fixedly installed on the top of the servo motor 1, a connecting column 2 is fixedly installed on the top of the fixed plate, a storage block 1 is fixedly installed on the top of the connecting column 2, a fixing groove 1 is provided on one side of the top of the storage block 1, slots 2 are provided on both sides of the inside of the storage block 1, a storage plate 1 is installed inside the slot 2, the slot 2 and the storage plate 1 are adapted to each other, the slots 2 and the storage plate 1 are provided with two identical ones, and the two slots 2 and the storage plate 1 are symmetrically distributed about the center line of the storage block 1.

[0016] Through the above technical solution, through the cooperation between slot two and storage plate one, when slot two is installed, storage plate one can be inserted into slot two, which can achieve rapid alignment and facilitate subsequent maintenance and replacement. In addition, the cooperation between the two slots two and storage plate one with respect to storage block one can place multiple test tubes during mixing, thereby improving the mixing efficiency.

[0017] Preferably, a second fixing groove is provided on the other side of the top of the first storage plate, a mounting ring is fixedly installed on the top of the second fixing groove, a second storage block is fixedly installed inside the mounting ring, and the mounting ring and the second storage block are adapted to each other.

[0018] Through the above technical solution, through the cooperation of the second fixing groove and the mounting ring, when mixing, mixing tubes of different diameters can be inserted into the mounting ring, which can improve the diversity of the mixing assembly and facilitate the placement of mixing tubes of different diameters.

[0019] Preferably, the stirring assembly includes a base, a second card slot is provided on the top of the base, a plug plate is installed in the second card slot, a third card slot is provided on the top of the plug plate, a placement plate is fixedly installed in the third card slot, a third fixing slot is provided on the top of the placement plate, a second servo motor is fixedly installed in the third fixing slot, a stirring rod is fixedly installed on the output shaft of the second servo motor, a limit block is provided on the base, a fourth fixing slot is provided on the top, a mounting hole one is provided on the left and right sides of the limit block, a fixing bolt three is provided on the outside of the plug plate, and the second card slot and the plug plate are adapted.

[0020] Through the above technical solution, through the second card slot and the plug plate, when installing the stirring component, the second card slot can be inserted into the plug plate, which can achieve rapid alignment of the two groups and facilitate subsequent installation.

[0021] Preferably, the fixed slot three is adapted to the servo motor two, the servo motor two and the stirring rod are located on the same vertical horizontal line as the fixed slot four, and the servo motor two, the stirring rod and the fixed slot four are in one-to-one correspondence.

[0022] Through the above technical scheme, through the cooperation of servo motor 2, stirring rod and fixed tank 4, the bacterial liquid after adding formalin is placed in the stirring tank, and the stirring tank is placed in the fixed tank 4. The servo motor 2 can be turned on to make the stirring rod rotate, so that the bacterial liquid inside the stirring tank placed in the fixed tank 4 can be stirred.

[0023] Preferably, the fixing bolt three passes through the base and the plug plate and extends to the interior of the fixing slot four, and the servo motor two, the stirring rod and the fixing slot four are provided with two identical ones, and the two servo motors two, the stirring rod and the fixing slot four are symmetrically distributed about the center line of the base.

[0024] Through the above technical solution, through the design of the fixing bolt three, when fixing the plug plate, it can also play a fixing role on the limit block, which can improve the connectivity between the plug plate and the limit block and the base to prevent them from being easily displaced.

[0025] Preferably, the detection component includes a detection live bacteria detector body, a fixing rod is fixedly installed on the top of the detection live bacteria detector body, a limit plate is fixedly installed on the outside of the fixing rod, a limit column is fixedly installed on the top of the limit plate, a top plate is arranged on the top of the limit plate, a mounting bolt is fixedly installed on the top of the top plate, a detection mirror is fixedly installed on the top of the limit plate, a positioning hole is opened on the outside of the detection mirror, the limiting column is adapted to the positioning hole, and the mounting bolt passes through the top plate and extends to the bottom of the limiting plate

[0026] Through the above technical solution and the design of the detection component, the culture medium body can be placed above the live bacteria chamber body, and then the bacterial count OD can be detected through the detection mirror. 600 and viable counts.

[0027] The present invention also provides a method for preparing an inactivated Edwardsiella tarda vaccine of turbot, comprising the following steps:

[0028] S1: Pick out Edwardsiella bacteria stored in the refrigerator with an inoculation needle and inoculate them into the culture medium respectively;

[0029] S2: After being placed at 30°C for 14-18 hours, a single colony is picked and placed in another culture medium 204. The bacterial solution in the culture medium 204 is then placed in a mixing tube, which is then inserted into the groove of the storage plate 1 509 above the mixing component 5 and the groove above the storage block 2 513. The temperature of the constant temperature culture body 301 is adjusted to 30°C by controlling the switch 303 and the display screen 302, and the speed of the servo motor 1 503 is controlled at 200 rpm. The culture is cultured until OD 600 =0.5~1.0, the inoculation amount is placed in another culture medium 204 at 0.5%~2%, and then the bacterial solution in the culture medium 204 is placed in the mixing tube, and then it is inserted into the groove of the storage plate 1 509 above the mixing component 5, and the groove above the storage block 2 513, and the temperature of the constant temperature culture body 301 is adjusted to 30°C by controlling the switch 303 and the display screen 302, and the speed of the servo motor 1 503 is controlled at 200rpm, and culture (8h~12h);

[0030] S3: When the bacterial concentration reaches about 15.0-60.0×10 8 CFU / mL, formalin was added to the culture medium with a final concentration of 0.05% (V / V);

[0031] S4: Then, the temperature of the constant temperature storage box body 6 is adjusted to 25° C., and the inactivation treatment is performed for 24 hours. The bacterial solution after adding formalin is placed in a stirring tank, and the stirring tank is placed in the fixed tank 4 710. Then, the stirring rod 708 is placed inside the stirring tank, and the bacterial solution is fully stirred once every 6 hours. After 24 hours, the inactivation is completed;

[0032] S5: Then adjust the concentration of the sterilization solution to 5.0-10.0×10 8 CFU / mL, and the turbot Edwardsiella tarda inactivated vaccine was obtained;

[0033] S6: The obtained turbot Edwardsiella tarda inactivated vaccine is packaged and placed in the refrigerator body 201 for storage, and the temperature of the refrigerator body 201 is adjusted to 4° C. for storage.

[0034] Compared with the prior art, the present invention provides a method and a device for preparing an inactivated Edwardsiella tarda vaccine for turbot, which have the following beneficial effects:

[0035] 1. The preparation method and preparation device of the turbot Edwardsiella tarda inactivated vaccine, when the bacterial count is 15.0-60.0×10 8 CFU / mL, 0.05% (V / V) formalin can be added for inactivation (usually the amount added is not less than 0.5%), reducing the amount of formalin added and reducing the stimulation of formalin to the fish body in the later stage; if the amount of formalin added is 0.5%, it needs to be washed with sterile saline several times and centrifuged after inactivation, which will increase the workload and increase the possibility of contamination by other bacteria;

[0036] 2. The preparation method and preparation device of the turbot Edwardsiella tarda inactivated vaccine, wherein the number of bacteria cultured by the device is 15.0-60.0×10 8 CFU / mL, the bacterial count can be reached in a short culture time, which shortens the culture time and improves work efficiency. In addition, the bacteria are in the logarithmic growth phase, which has advantages in all aspects.

[0037] 3. The preparation method and preparation device of the turbot Edwardsiella tarda inactivated vaccine can improve the immune protection rate as a whole, which is higher than the immune effect of conventional antigens. The immune protection rate can reach 85% after 28 days of immunization. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of the three-dimensional structure of the present invention;

[0039] Figure 2 This is a schematic diagram of the disassembly of the detection components of the present invention;

[0040] Figure 3 This is a schematic diagram of the rotating structure of the storage assembly of the present invention;

[0041] Figure 4 This is a schematic diagram of the rotating structure of the constant temperature culture component of the present invention;

[0042] Figure 5 This is a schematic diagram of the split structure of the storage tank and the mounting plate of the present invention;

[0043] Figure 6 This is a schematic diagram of the installation structure of the constant temperature storage box body and the stirring assembly of the present invention;

[0044] Figure 7 This is a schematic diagram of the split structure of the detection component of the present invention;

[0045] Figure 8 This is a schematic diagram of the split structure of the mixing component of the present invention;

[0046] Fig. 9 This is a schematic diagram of the split structure of the stirring assembly of the present invention;

[0047] Fig.10 This is a schematic diagram of the installation structure of the constant temperature culture component and the mixing component of the present invention;

[0048] Fig.11 The present invention is a flow chart of the preparation method.

[0049] Among them: 1. Bottom plate; 2. Storage component; 201. Refrigerator body; 202. Storage bar; 203. Through slot; 204. Culture medium body; 3. Constant temperature culture component; 301. Constant temperature incubator body; 302. Display screen; 303. Control switch; 304. Divider bar; 305. Storage slot; 306. Card slot 1; 307. Mounting plate; 308. Fixing bolt 1; 4. Detection component; 401. Mounting seat; 402. Mounting slot; 403. Slot 1; 404. Sealing pad; 405. Fixing bolt 2; 406. Connecting column 1; 407. Probe; 408. Temperature detector body; 5. Mixing component; 501. Protective cover; 502. Protective cover; 503. Servo motor 1; 504. Fixing plate; 505. Connecting column 2 ; 506, storage block one; 507, fixed slot one; 508, slot two; 509, storage plate one; 510, fixed slot two; 512, mounting ring; 513, storage block two; 6, constant temperature storage box body; 7, stirring assembly; 701, base; 702, slot two; 703, plug plate; 704, slot three; 705, placement plate; 706, fixed slot three; 707, servo motor two; 708, stirring rod; 709, limit block; 710, fixed slot four; 711, mounting hole one; 712, fixing bolt three; 8, detection assembly; 801, detection live bacteria detector body; 802, fixing rod; 803, limit plate; 804, limit column; 805, top plate; 806, mounting bolt; 807, detection mirror; 808, positioning hole. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in 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.

[0051] Embodiment 1: Figure 1-11As shown, a preparation device for an inactivated Edwardsiella tarda vaccine of turbot provided by the present invention comprises a bottom plate 1, a storage component 2 is fixedly installed on the top of the bottom plate 1, a constant temperature culture component 3 is fixedly installed on the top of the bottom plate 1, a detection component 4 is fixedly installed on the top of the constant temperature culture component 3, a mixing component 5 is fixedly installed inside the constant temperature culture component 3, a constant temperature storage box body 6 is fixedly installed on the top of the bottom plate 1, a stirring component 7 is fixedly installed inside the constant temperature storage box body 6, and a detection component 8 is fixedly installed on the top of the bottom plate 1;

[0052] The storage assembly 2 includes a refrigerator body 201 , in which a storage column 202 is installed and inserted. A through slot 203 is provided on the top of the storage column 202 , and a culture medium body 204 is provided on the top of the storage column 202 .

[0053] Specifically, the constant temperature incubation component 3 includes a constant temperature incubator body 301, a display screen 302 is arranged on the front side of the constant temperature incubator body 301, a control switch 303 is arranged on the front side of the constant temperature incubator body 301, a partition bar 304 is fixedly installed inside the constant temperature incubator body 301, a storage slot 305 is opened at the bottom of the constant temperature incubator body 301, a card slot 306 is opened at the bottom of the constant temperature incubator body 301, a mounting plate 307 is arranged inside the storage slot 305, and a fixing bolt is fixedly installed on the top of the mounting plate 307. 1 308, the constant temperature incubator body 301 is plug-connected with the mounting plate 307 through the storage slot 305 and the card slot 1 306, and the fixing bolt 1 308 passes through the mounting plate 307 and extends to the interior of the constant temperature incubator body 301. The advantage is that the storage slot 305 and the card slot 1 306 cooperate with the mounting plate 307, so that the mounting plate 307 can be quickly aligned during installation. Then, the fixing bolt 1 308 is rotated to achieve its quick installation, which can facilitate the maintenance and repair of the servo motor 503 in the later stage.

[0054] Specifically, the detection component 4 includes a mounting seat 401, a mounting groove 402 is provided on the inner side of the mounting seat 401, a slot 1 403 is provided on the inner side of the mounting seat 401, a sealing gasket 404 is fixedly installed on the top of the mounting seat 401, a fixing bolt 2 405 is arranged on the top of the mounting seat 401, a connecting column 1 406 is plugged and installed inside the slot 1 403, a probe 407 is fixedly installed at the bottom of the connecting column 1 406, a temperature detector body 408 is fixedly installed on the top of the connecting column 1 406, the probe 407 penetrates the mounting seat 401 and extends to the interior of the constant temperature incubator body 301, and the detection component 4 is located at the top of the constant temperature incubator body 301. The advantage is that through the design of the detection component 4, when the bacterial solution is cultured at a constant temperature, the probe 407 can be extended into the interior of the constant temperature incubator body 301 to detect the internal temperature, and then the detected data is transmitted to the surface of the temperature detector body 408 for display, so that the staff can understand the production temperature of the bacterial solution.

[0055] Embodiment 2: Figure 2-11 As shown, as an improvement to the previous embodiment.

[0056] Specifically, the mounting seat 401 is matched with the connecting column 406 through the slot 403, the sealing gasket 404 is a rubber sealing gasket, and the fixing bolt 405 passes through the mounting seat 401 and extends to the interior of the constant temperature incubator body 301. The advantage is that through the cooperation between the mounting seat 401, the slot 403 and the connecting column 406, when installing the components above the connecting column 406, the connecting column 406 can be inserted through the slot 403, which can play a role in rapid alignment, and with the sealing gasket 404, it can prevent bacteria from entering the interior, resulting in the sterile environment of the bacteria being affected, affecting the normal growth of the bacteria.

[0057] Specifically, the mixing component 5 includes a protective cover 501, a protective cover 502 is plugged into the top of the protective cover 501, a servo motor 1 503 is fixedly installed inside the constant temperature incubator body 301, a fixing plate 504 is fixedly installed on the top of the servo motor 1 503, a connecting column 2 505 is fixedly installed on the top of the fixing plate 504, a storage block 1 506 is fixedly installed on the top of the connecting column 2 505, a fixing groove 1 507 is opened on one side of the top of the storage block 1 506, two slots 508 are opened on both sides of the storage block 1 506, a storage plate 1 509 is plugged into the slot 2 508, and a fixing groove 1 507 is opened on one side of the top of the storage block 1 506, and a fixing groove 1 508 is opened on both sides of the storage block 1 506. 08 is compatible with the storage plate 1 509, and the slot 2 508 and the storage plate 1 509 are provided with the same two, and the two slots 2 508 and the storage plate 1 509 are symmetrically distributed about the center line of the storage block 1 506. The advantage is that through the cooperation of the slot 2 508 and the storage plate 1 509, when the slot 2 508 is installed, the storage plate 1 509 can be inserted into the slot 2 508, so that it can be quickly aligned, which is convenient for subsequent maintenance and replacement, and the cooperation of the two slots 2 508 and the storage plate 1 509 with respect to the storage block 1 506 can place multiple test tubes during mixing, so as to improve the mixing efficiency.

[0058] Specifically, a fixing groove 2 510 is opened on the other side of the top of the storage plate 1 509, and a mounting ring 512 is fixedly installed on the top of the fixing groove 2 510, and a storage block 2 513 is fixedly installed inside the mounting ring 512. The mounting ring 512 and the storage block 2 513 are adapted to each other. The advantage is that, through the cooperation of the fixing groove 2 510 and the mounting ring 512, mixing tubes of different diameters can be inserted into the mounting ring 512 during mixing, which can improve the diversity of the mixing component 5 and facilitate the placement of mixing tubes of different diameters.

[0059] Embodiment 3: Figure 3-11 As shown, as an improvement to the previous embodiment.

[0060] Specifically, the stirring assembly 7 includes a base 701, a second card slot 702 is provided on the top of the base 701, a plug plate 703 is installed in the second card slot 702, a third card slot 704 is provided on the top of the plug plate 703, a placement plate 705 is fixedly installed in the third card slot 704, a fixing slot 3 706 is provided on the top of the placement plate 705, a servo motor 2 707 is fixedly installed in the fixing slot 3 706, a stirring rod 708 is fixedly installed on the output shaft of the servo motor 2 707, a limiting block 709 is provided on the base 701, a fixing slot 4 710 is provided on the top, mounting holes 1 711 are provided on the left and right sides of the limiting block 709, a fixing bolt 3 712 is provided on the outer side of the plug plate 703, the second card slot 702 and the plug plate 703 are adapted to each other, and the advantage is that through the second card slot 702 and the plug plate 703, when installing the stirring assembly 7, the plug plate 703 can be inserted into the plug plate 703 through the second card slot 702, so that the two groups can be quickly aligned, which is convenient for subsequent installation.

[0061] Specifically, the fixed tank three 706 and the servo motor two 707 are adapted to each other, the servo motor two 707 and the stirring rod 708 are located on the same vertical horizontal line as the fixed tank four 710, and the servo motor two 707, the stirring rod 708 and the fixed tank four 710 are in one-to-one correspondence. The advantage is that, through the cooperation of the servo motor two 707, the stirring rod 708 and the fixed tank four 710, the bacterial liquid after adding formalin is placed in the stirring tank, and the stirring tank is placed in the fixed tank four 710. The servo motor two 707 can be turned on to rotate the stirring rod 708, and the bacterial liquid inside the stirring tank placed in the fixed tank four 710 can be stirred.

[0062] Specifically, the fixing bolt three 712 passes through the base 701 and the plug plate 703 and extends to the interior of the fixing groove four 710. The servo motor two 707, the stirring rod 708 and the fixing groove four 710 are provided with two identical ones. The two servo motors two 707, the stirring rod 708 and the fixing groove four 710 are symmetrically distributed about the center line of the base 701. The advantage is that through the design of the fixing bolt three 712, when fixing the plug plate 703, the limit block 709 can be fixed at the same time, which can improve the connectivity between the plug plate 703 and the limit block 709 and the base 701 to prevent them from being easily displaced.

[0063] Specifically, the detection component 8 includes a detection live bacteria detector body 801, a fixing rod 802 is fixedly installed on the top of the detection live bacteria detector body 801, a limiting plate 803 is fixedly installed on the outer side of the fixing rod 802, a limiting column 804 is fixedly installed on the top of the limiting plate 803, a top plate 805 is arranged on the top of the limiting plate 803, a mounting bolt 806 is fixedly installed on the top of the top plate 805, a detection mirror 807 is fixedly installed on the top of the limiting plate 803, a positioning hole 808 is opened on the outer side of the detection mirror 807, the limiting column 804 is adapted to the positioning hole 808, and the mounting bolt 806 passes through the top plate 805 and extends to the bottom of the limiting plate 803. The advantage is that, through the design of the detection component 8, the culture medium body 204 can be placed above the live bacteria detector body 801, and then the bacterial count OD is detected through the detection mirror 807. 600 and viable counts.

[0064] A method for preparing an inactivated Edwardsiella tarda vaccine for turbot comprises the following steps:

[0065] S1: The Edwardsiella bacteria stored in the refrigerator body 201 are picked out with an inoculation needle and inoculated into the culture medium body 204 respectively;

[0066] S2: After being placed at 30°C for 14-18 hours, a single colony is picked and placed in another culture medium 204. The bacterial solution in the culture medium 204 is then placed in a mixing tube, which is then inserted into the groove of the storage plate 1 509 above the mixing component 5 and the groove above the storage block 2 513. The temperature of the constant temperature culture body 301 is adjusted to 30°C by controlling the switch 303 and the display screen 302, and the speed of the servo motor 1 503 is controlled at 200 rpm. The culture is cultured until OD 600 =0.5~1.0, the inoculation amount is placed in another culture medium 204 at 0.5%~2%, and then the bacterial solution in the culture medium 204 is placed in the mixing tube, and then it is inserted into the groove of the storage plate 1 509 above the mixing component 5, and the groove above the storage block 2 513, and the temperature of the constant temperature culture body 301 is adjusted to 30°C by controlling the switch 303 and the display screen 302, and the speed of the servo motor 1 503 is controlled at 200rpm, and culture (8h~12h);

[0067] S3: When the bacterial concentration reaches about 15.0-60.0×10 8 CFU / mL, formalin was added to the bacterial solution in medium 204 to a final concentration of 0.05% (V / V);

[0068] S4: Then, the temperature of the constant temperature storage box body 6 is adjusted to 25° C., and the inactivation treatment is performed for 24 hours. The bacterial solution after adding formalin is placed in a stirring tank, and the stirring tank is placed in the fixed tank 4 710. Then, the stirring rod 708 is placed inside the stirring tank, and the bacterial solution is fully stirred once every 6 hours. After 24 hours, the inactivation is completed;

[0069] S5: Then adjust the concentration of inactivated vaccine to 5.0-10.0×10 8 CFU / mL, and the turbot Edwardsiella tarda inactivated vaccine was obtained;

[0070] S6: The obtained turbot Edwardsiella tarda inactivated vaccine is packaged and placed in the refrigerator body 201 for storage, and the temperature of the refrigerator body 201 is adjusted to 4° C. for storage.

[0071] Comparison of inactivation effects: The turbot Edwardsiella tarda was cultured to a concentration of about 60.0×10 8 CFU / mL, add formalin to the bacterial solution to a final concentration of 0.5% (V / V) or 0.05% (V / V), inactivate at 25°C for 24h, 48h and 168h, and take samples to test the inactivation. Take 0.2mL of the inactivated bacterial solution and inoculate it into LB solid medium, culture at 30°C for 48 hours. If there is no sterile growth, it can be judged as qualified for inactivation, otherwise it is unqualified.

[0072]

[0073] 60 healthy turbots were randomly divided into experimental group A, experimental group B, experimental group C, experimental group D and control group E, with 20 turbots in each group. Group A and group B were immunized with different concentrations of inactivated vaccines (0.1 mL / turbot) made from Edwardsiella tarda harvested by conventional culture, group C and group D were immunized with different concentrations of inactivated vaccines (0.1 mL / turbot) made from Edwardsiella tarda harvested by patented culture, and group C was injected with 0.85% sterile saline (0.1 mL / turbot). After immunization and injection, the test fish in each group were routinely raised. The number of days from immunization and injection to artificial infection test was 28 days. After 28 days, turbot Edwardsiella was artificially infected by intramuscular injection, and the bacterial concentration was adjusted to about 4×10 6 CFU / mL, 0.1mL / tail, after artificial infection injection, each group of turbot was routinely fed and observed for 14 consecutive days, and the incidence and death of the tested fish were recorded every day. The vaccine protection rate was calculated according to the following formula.

[0074] Vaccine protection rate = (mortality rate of control group - mortality rate of immunized group) / mortality rate of control group × 100%. The results are as follows: The fifth day after artificial infection was the peak period of death of turbot in each test group and control group. The mortality rate of control group E within 14 days was 100%, and the mortality rates of test groups A and B within 14 days were 25% and 20% respectively; the mortality rates of test groups C and D within 14 days were both 15%. The Edwardsiella tarda obtained by the patented culture method has good immunogenicity, and the immune protection rate of turbot is 85%, which is higher than the 75-80% of the conventional culture method.

[0075]

[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A device for preparing an inactivated Edwardsiella tarda vaccine for turbot, comprising a bottom plate (1) and characterized in that: A storage component (2) is fixedly mounted on the top of the bottom plate (1), a constant temperature culture component (3) is fixedly mounted on the top of the bottom plate (1), a detection component (4) is fixedly mounted on the top of the constant temperature culture component (3), a mixing component (5) is fixedly mounted inside the constant temperature culture component (3), a constant temperature storage box body (6) is fixedly mounted on the top of the bottom plate (1), a stirring component (7) is fixedly mounted inside the constant temperature storage box body (6), and a detection component (8) is fixedly mounted on the top of the bottom plate (1); The storage assembly (2) comprises a refrigerator body (201), a storage column (202) is plugged and installed inside the refrigerator body (201), a through slot (203) is provided through the top of the storage column (202), and a culture medium body (204) is provided on the top of the storage column (202).

2. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 1, characterized in that: The constant temperature incubation component (3) comprises a constant temperature incubator body (301), a display screen (302) is arranged on the front side of the constant temperature incubator body (301), a control switch (303) is arranged on the front side of the constant temperature incubator body (301), a partition bar (304) is fixedly installed inside the constant temperature incubator body (301), a storage slot (305) is provided at the bottom of the constant temperature incubator body (301), a card slot 1 (306) is provided at the bottom of the constant temperature incubator body (301), a mounting plate (307) is arranged inside the storage slot (305), a fixing bolt 1 (308) is fixedly installed on the top of the mounting plate (307), the constant temperature incubator body (301) is plug-connected with the mounting plate (307) through the storage slot (305) and the card slot 1 (306), and the fixing bolt 1 (308) passes through the mounting plate (307) and extends to the inside of the constant temperature incubator body (301).

3. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 2, characterized in that: The detection component (4) comprises a mounting seat (401), a mounting groove (402) is provided on the inner side of the mounting seat (401), a slot 1 (403) is provided on the inner side of the mounting seat (401), a sealing gasket (404) is fixedly installed on the top of the mounting seat (401), a fixing bolt 2 (405) is arranged on the top of the mounting seat (401), a connecting column 1 (406) is plugged and installed inside the slot 1 (403), a probe (407) is fixedly installed on the bottom of the connecting column 1 (406), a temperature detector body (408) is fixedly installed on the top of the connecting column 1 (406), the probe (407) passes through the mounting seat (401) and extends to the inside of the constant temperature incubator body (301), and the detection component (4) is located on the top of the constant temperature incubator body (301).

4. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 3, characterized in that: The mounting seat (401) is matched with the connecting column (406) through the slot (403), the sealing gasket (404) is a rubber sealing gasket, and the fixing bolt (405) passes through the mounting seat (401) and extends to the interior of the constant temperature incubator body (301).

5. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 4, characterized in that: The mixing component (5) comprises a protective cover (501), a protective cap (502) is plugged into the top of the protective cover (501), a servo motor (503) is fixedly installed inside the constant temperature incubator body (301), a fixing plate (504) is fixedly installed on the top of the servo motor (503), a connecting column (505) is fixedly installed on the top of the fixing plate (504), a storage block (506) is fixedly installed on the top of the connecting column (505), and the storage block (506) is fixedly installed on the top of the storage block (506). 06) A fixing groove 1 (507) is provided on one side of the top, and slots 2 (508) are provided on both sides inside the storage block 1 (506), and a storage plate 1 (509) is installed inside the slot 2 (508), and the slot 2 (508) and the storage plate 1 (509) are adapted to each other, and the slots 2 (508) and the storage plate 1 (509) are provided with two identical ones, and the two slots 2 (508) and the storage plate 1 (509) are symmetrically distributed about the center line of the storage block 1 (506).

6. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 1, characterized in that: A second fixing groove (510) is provided on the other side of the top of the first storage plate (509), a mounting ring (512) is fixedly installed on the top of the second fixing groove (510), a second storage block (513) is fixedly installed inside the mounting ring (512), and the mounting ring (512) and the second storage block (513) are adapted to each other.

7. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 1, characterized in that: The stirring assembly (7) comprises a base (701), a second card slot (702) is provided on the top of the base (701), a plug board (703) is installed in the second card slot (702), a third card slot (704) is provided on the top of the plug board (703), a placement board (705) is fixedly installed in the third card slot (704), a third fixed slot (706) is provided on the top of the placement board (705), and a third fixed slot (706) is fixed in the third fixed slot (706). A servo motor 2 (707) is fixedly installed, a stirring rod (708) is fixedly installed on the output shaft of the servo motor 2 (707), a limit block (709) is provided on the base (701), a fixing groove 4 (710) is provided on the top, a mounting hole 1 (711) is provided on the left and right sides of the limit block (709), a fixing bolt 3 (712) is provided on the outside of the plug plate (703), and the slot 2 (702) and the plug plate (703) are adapted to each other.

8. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 1, characterized in that: The fixed slot three (706) and the servo motor two (707) are adapted to each other, the servo motor two (707) and the stirring rod (708) are located on the same vertical horizontal line as the fixed slot four (710), and the servo motor two (707), the stirring rod (708) and the fixed slot four (710) are in one-to-one correspondence.

9. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 1, characterized in that: The fixing bolt three (712) passes through the base (701) and the plug plate (703) and extends to the interior of the fixing slot four (710). The servo motor two (707), the stirring rod (708) and the fixing slot four (710) are provided with two identical ones. The two servo motors two (707), the stirring rod (708) and the fixing slot four (710) are symmetrically distributed about the center line of the base (701).

10. The device for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claim 1, characterized in that: The detection assembly (8) comprises a detection live bacteria detector body (801), a fixing rod (802) is fixedly installed on the top of the detection live bacteria detector body (801), a limiting plate (803) is fixedly installed on the outer side of the fixing rod (802), a limiting column (804) is fixedly installed on the top of the limiting plate (803), a top plate (805) is arranged on the top of the limiting plate (803), a mounting bolt (806) is fixedly installed on the top of the top plate (805), a detection mirror (807) is fixedly installed on the top of the limiting plate (803), a positioning hole (808) is opened on the outer side of the detection mirror (807), the limiting column (804) is adapted to the positioning hole (808), and the mounting bolt (806) passes through the top plate (805) and extends to the bottom of the limiting plate (803).

11. The method for preparing an inactivated Edwardsiella tarda vaccine of turbot according to claims 1-10, characterized in that: The following steps are involved: S1: Edwardsiella tarda of turbot refrigerated in the refrigerator body (201) is picked out with an inoculation needle and inoculated into the culture medium body (204) respectively; S2: After being placed at 30°C for 14 to 18 hours, a single colony is picked and placed in another culture medium (204), and then the bacterial solution in the culture medium (204) is placed in a mixing tube, which is then inserted into the groove of the storage plate 1 (509) above the mixing component (5) and the groove above the storage block 2 (513). The temperature of the constant temperature culture body (301) is adjusted to 30°C by controlling the switch (303) and the display screen (302), and the speed of the servo motor 1 (503) is controlled at 200 rpm. The culture is continued until OD 600 =0.5~1.0, the inoculation amount is placed in another culture medium (204) at 0.5%~2%, and then the bacterial solution in the culture medium (204) is placed in a mixing tube, which is then inserted into the groove of the storage plate 1 (509) above the mixing component (5) and the groove above the storage block 2 (513), and the temperature of the constant temperature culture body (301) is adjusted to 30°C by controlling the switch (303) and the display screen (302), and the speed of the servo motor 1 (503) is controlled at 200rpm, and the culture is carried out (8h~12h); S3: When the bacterial concentration reaches about 15.0-60.0×10 8 CFU / mL, formalin was added to the bacterial solution in the culture medium (204) to a final concentration of 0.05% (V / V); S4: The temperature of the thermostatic storage box body (6) is then adjusted to 25°C, and an inactivation treatment is performed for 24 hours. The bacterial solution after adding formalin is placed in a stirring tank, and the stirring tank is placed in the fixed tank four (710). Then, the stirring rod (708) is located inside the stirring tank, and the bacterial solution is fully stirred once every 6 hours. After 24 hours, the inactivation is completed; S5: Then adjust the concentration of the sterilization solution to 5.0-10.0×10 8 CFU / mL, and the turbot Edwardsiella tarda inactivated vaccine was obtained; S6: The obtained turbot Edwardsiella tarda inactivated vaccine is packaged and placed in a refrigerator body (201) for storage, and the temperature of the refrigerator body (201) is adjusted to 4°C for storage.

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