CN117159695A - Vaccine based on aloe as carrier and application of vaccine in aquatic animals - Google Patents
Vaccine based on aloe as carrier and application of vaccine in aquatic animals Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention belongs to the technical field of aquatic products, and particularly relates to a vaccine based on aloe as a carrier and application of the vaccine in aquatic animals, one of the purposes of the invention is to provide the application of aloe as a vaccine carrier in aquatic animals, the resistance of bullfrog and channel catfish soaked by the aloe carrier inactivated vaccine to corresponding pathogenic infection is obviously enhanced, a higher serum antibody titer is maintained in vivo, lgM gene expression is also very high, the method is simple and efficient or easy to popularize, the invention also provides a test method of the aloe and vaccine carrier in the bullfrog or channel catfish, the test application method of the invention has outstanding evaluation effect, the immune effect is better, and the invention is used as a natural plant, has no toxic effect on fish bodies, people and environment, and has no risk of drug residue.
Description
Technical Field
The invention belongs to the technical field of aquatic products, and particularly relates to application of aloe serving as a vaccine carrier in aquatic animals.
Background
Aloe is one of the few edible species in the genus aloe, and its products are widely used in the fields of foods, cosmetics, health care, medicine, etc. The aloe has the effects of sterilizing, anti-inflammatory, moistening, caring skin, invigorating stomach, relieving diarrhea, tonifying heart, promoting blood circulation, immunizing, regenerating, immunity, resisting tumor, removing toxic substance, resisting aging, relieving pain, tranquilizing, and preventing sunburn. There is no report of application research in aquatic animals.
Yersinia ruckeri is a gram negative bacterium, is the pathogen of fish enteritis red mouth disease (ERM), is a serious fish disease popular in the world water-borne aquaculture industry, and mainly damages objects such as rainbow trout, sturgeon, channel catfish and the like, wherein the popular water temperature is 15-22 ℃, and the harm is huge. Pathological changes in diseased channel catfish have shown that sepsis is common to most organs (including kidneys, spleen, liver and gastrointestinal tract) and is accompanied by inflammation. During infection, yersinia ruckeri may invade the gill epithelium and gastrointestinal epithelium early, then enter the blood circulation later, then further infect the spleen and dry kidneys, and accumulate in lymphoid organs, eventually destroying the immune system, leading to body death. At present, antibiotics are mainly used for prevention and treatment, but the prevention and treatment effect is poor. In addition, because the water temperature is very low when the disease is popular, the fish does not ingest and can not be treated by an external medicine, and disease resistance is carried out by completely utilizing autoimmunity, the development of a vaccine becomes an important mode for preventing the disease.
At present, the infection of the Isaria sedge is found in various farmed fishes such as tilapia, clarias, acipenser sinensis and the like, so that great economic loss is caused, especially the species continuation of the Acipenser sinensis is influenced, and the Acipenser sinensis is worthy of important attention. In cultured amphibians, infection with Elizabeth bacteria has also been found in several species, such as tiger frog, xenopus laevis, frog, bullfrog, black spot frog, thorn chest frog, etc. The cultured amphibians infected with the Elizabeth bacteria can show obvious typical symptoms such as head distortion, head breaking, white eyes and the like. The Irish white fungus infected by frog is mainly Irish white fungus of Michelia milbeum. Histopathological studies show that the melaleuca millixiviata can cause damage to organs and tissues of the whole body, the damage to tissues and organs such as brain, intestinal tract, liver, spleen, kidney and the like, the main pathological changes are denaturation, necrosis and inflammatory reaction, and the dysfunction of tissues and organs causes death of the infected frog. The nerve cells of the brain infected with the frog are injured and have inflammatory infiltration, and the Mier Elizabeth fungus possibly penetrates through the blood brain barrier to enter the brain, so that the brain of the frog is seriously damaged, and the frog clinically appears as head distortion, head breakage and white eyes. Since the Mier Elizabeth fungus can break through the blood brain barrier, so that the medicine is difficult to reach and kill, and the Mier Elizabeth fungus has the possibility of vertical transmission, the primary prevention and control measure of the Mier Elizabeth fungus disease is to develop a vaccine for prevention.
Because aquatic animals are difficult to vaccinate by injection, and no breakthrough is realized in oral vaccines at present, soaking the vaccine becomes a direction worthy of important research.
Disclosure of Invention
In order to solve any of the above problems, the present invention provides a method for preparing a vector based on aloe and vaccine: the method comprises the following steps: firstly, inoculating bacteria into a culture medium, and shake culturing; secondly, inactivating the bacterial liquid to prepare an inactivated bacterial vaccine, and detecting whether viable bacteria exist or not to evaluate the inactivation effect; finally, the inactivated vaccine is mixed with aloe and diluted to form the carrier based on aloe and vaccine.
Further, the bacteria are Elizabeth milbemyces, the culture medium is BHI, the shake culture is performed for 10-18 hours at 25-28 ℃, the inactivated bacteria are whole inactivated bacteria, the detection is performed by using a flat plate experiment, the mixing is uniformly mixed with aloe supernatant in a ratio of 1:1, and the dilution is performed to 20% concentration.
Further, formaldehyde is used as the fire extinguishing bacteria.
The invention also provides an application method of the vector based on aloe and vaccine in aquatic animals, which comprises the following steps: randomly grouping aquatic animals, and respectively arranging an aloe soaking group, an aloe vector inactivated vaccine injection group, an aloe vector inactivated vaccine soaking group, an inactivated vaccine soaking group and an inactivated vaccine injection group; and a second step of: soaking or injecting according to groups; and a third step of: detecting gene expression and detecting antibody titer.
Furthermore, after injection according to groups, bacterial infection experiments are also required to be carried out after the injection, and the state or morbidity and mortality of aquatic animals are observed.
Further, the detection gene expression is that blood of the aquatic animal is taken on the X-th day, total RNA is extracted, cDNA is inverted to be used as a qPCR template, and lgM gene expression is detected.
Further, the titer of the detection antibody is that blood of each group of aquatic animals is taken in week X, supernatant is collected by centrifugation, and serum antibodies are detected by an indirect ELISA method.
Further, the aquatic animal is bullfrog or channel catfish.
Further, after the injection is performed in groups, an infection experiment is performed on the Ictalurus punctatus by injecting Yersinia ruckeri into the abdominal cavity, and the state and the morbidity and mortality of the Ictalurus punctatus are observed and recorded.
The invention also provides a vector based on aloe and a vaccine, comprising aloe or a vaccine.
Compared with the prior art, the invention has one of the following advantages and beneficial effects
The preparation method is simple and efficient or popularized, the evaluation effect of the test application method is outstanding, the absorption effect is good, the immune effect is better, and the test application method is used as a natural plant, has no toxic effect on fish bodies, people and the environment and has no risk of drug residues.
Drawings
FIG. 1 effect of different vaccines on Bullfrog infection with Elizabeth milbezoar;
FIG. 2 effect of different vaccines on Yersinia ruckeri infection with channel catfish;
FIG. 3 effect of different vaccines on IgM gene expression in bullfrog blood;
FIG. 4 effect of different vaccines on IgM gene expression in channel catfish blood;
FIG. 5 effect of different vaccines on antibody levels in bullfrog serum;
FIG. 6 effect of different vaccines on antibody levels in channel catfish serum.
Detailed Description
The invention aims at providing the application of aloe serving as a vaccine carrier in aquatic animals, wherein the resistance of bullfrog and channel catfish soaked by an aloe carrier inactivated vaccine to corresponding pathogenic infection is obviously enhanced, a higher serum antibody titer is maintained in vivo, and lgM gene expression is also high.
Embodiment one
The polysaccharides, amino acids and organic acids contained in aloe can form natural moisturizing factors, and can be directly absorbed by skin to play a role in skin care. The aloe has remarkable moisturizing effect on skin, has high compatibility with human body, strong skin penetrability, is easy to be absorbed by skin, is safe and non-irritating, is an efficacy additive with strong safety worthy of further development, can be widely applied to dermatology, and can be used as an excellent moisturizing cosmetic efficacy additive or an external skin xerosis preparation additive. Therefore, the vaccine is developed into a carrier of aquatic animal vaccine, and the vaccine is dissolved in aloe water to be easily sprayed or soaked in fish, so that the vaccine is absorbed into aquatic animal bodies together with aloe by skin, and the result of vaccination is achieved.
The aloe has the characteristics of nature, no toxic or side effect and multiple biological functions, is an ideal natural medicine, and can be widely used for various aquatic animal vaccine carriers.
The aloe used as a vaccine carrier and the application related method in aquatic animals are described as follows: a preparation method based on aloe and vaccine as carrier comprises inoculating bacteria into culture medium, shake culturing; secondly, inactivating the bacterial liquid to prepare an inactivated bacterial vaccine, and detecting whether viable bacteria exist or not to evaluate the inactivation effect; finally, the inactivated vaccine is mixed with aloe and diluted to form the carrier based on aloe and vaccine.
The survival rate of the bullfrog infected with the Mier Elizabeth can be effectively improved after the bullfrog is soaked by the aloe carrier inactivated vaccine (Mier Elizabeth), and the survival rate of immune means such as injection of the aloe carrier inactivated vaccine (Mier Elizabeth), injection of the inactivated vaccine (Mier Elizabeth), soaking of the inactivated vaccine (Mier Elizabeth) and the like is higher than that of the aloe carrier inactivated vaccine (Mier Elizabeth); the survival rate of the channel catfish infected by the yersinia ruckeri can be effectively improved after the channel catfish is soaked by the aloe carrier inactivated vaccine (yersinia ruckeri), and the survival rate of the immune means such as injection of the aloe carrier inactivated vaccine (yersinia ruckeri), injection of the inactivated vaccine (yersinia ruckeri), soaking of the inactivated vaccine (yersinia ruckeri) and the like is higher than that of the aloe carrier inactivated vaccine (yersinia ruckeri). Soaking bullfrog in aloe vector inactivated vaccine (Mier Elizabeth fungus), gradually increasing antibody titer in serum for 1-8 weeks, and regulating expression of IgM gene in blood for 1-14d, so that the expression of the detection gene is that blood of aquatic animals such as 1, 3, 5, 7, 14 and 28d is taken on day X, total RNA is extracted, and the total RNA is reversed to cDNA as qPCR template, and lgM gene expression condition is detected; the titer of the detection antibody is that blood of each group of aquatic animals is taken at week X, such as weeks 1, 2, 4 and 8, supernatant is collected by centrifugation, and serum antibodies are detected by an indirect ELISA method.
Compared with the methods of injection of an aloe vector inactivated vaccine (Mier Elizabeth fungus), injection of an inactivated vaccine (Mier Elizabeth fungus), soaking of an inactivated vaccine (Mier Elizabeth fungus) and the like, the antibody titer is higher, and IgM gene expression in blood is obviously up-regulated; the antibody titer of the aloe vector inactivated vaccine (yersinia ruckeri) soaked in the channel catfish in serum is gradually increased for 1-8 weeks, the expression of IgM genes in blood is up-regulated all the time, and compared with the mode of injecting the aloe vector inactivated vaccine (yersinia ruckeri), injecting the inactivated vaccine (yersinia ruckeri), soaking the inactivated vaccine (yersinia ruckeri) and the like, the antibody titer is higher, and the IgM gene expression in blood is obviously up-regulated.
Second embodiment
In the first embodiment, unlike the first embodiment, the following description is given of bullfrog as an aquatic object:
influence of different vaccines on Bullfrog infected with Mier Elizabeth fungus, the Mier Elizabeth fungus related to the invention is stored by a fishing medicine laboratory of the China aquatic research institute of aquatic products. Mashing aloe (prepared from purchased plant), dissolving, removing residues, and keeping supernatant at 4deg.C.
The method comprises the steps of inoculating the illisha millariella mellea into a BHI culture medium, performing shake culture at 25-28 ℃, preferably 28 ℃ for 18 hours, inactivating bacterial liquid by using 0.5% formaldehyde to prepare an inactivated whole bacterial vaccine, and detecting whether viable bacteria exist or not by using a flat plate experiment to evaluate the inactivation effect. Mixing the inactivated vaccine with aloe supernatant at a ratio of 1:1, and diluting to 20%. The bullfrog is randomly grouped, 30 bullfrog groups are respectively provided with an aloe soaking group (A), an aloe vector inactivated vaccine injection group (B), an aloe vector inactivated vaccine soaking group (C), an inactivated vaccine soaking group (D) and an inactivated vaccine injection group (E), and the grouping mode is a treatment mode. After 2 weeks of vaccine injection, the bullfrog was subjected to an infection experiment by injecting Elizabeth mile into the abdominal cavity, and the state and morbidity and mortality of the bullfrog were observed and recorded. The experiment is carried out in black large barrels with covers, each group of barrels is provided with water level reaching the frog neck, the water temperature is preferably 26+/-2 ℃, and the water is normally fed during the experiment. The study result is shown in figure 1, the survival number of the bullfrog in group C is the highest, all the bullfrog in group A and group D die, and part of the bullfrog in group BE survives, which shows that the soaking treatment mode of the aloe carrier inactivated vaccine is the most effective.
The bullfrog is randomly grouped, 30 bullfrog groups are respectively provided with an aloe soaking group (A), an aloe vector inactivated vaccine injection group (B), an aloe vector inactivated vaccine soaking group (C), an inactivated vaccine soaking group (D) and an inactivated vaccine injection group (E), and the grouping mode is a treatment mode. The bullfrog experiment is carried out in black large barrels with covers, each group of barrels is provided with water level reaching the frog neck, the water temperature is preferably 26+/-2 ℃, and the bullfrog experiment is normally fed during the experiment. Taking various frogs for blood drawing, extracting total RNA, reversing the total RNA into cDNA as qPCR template, and detecting lgM gene expression condition, wherein the primers are shown in table 1; the frog blood of each group is extracted at 1, 2, 4 and 8 weeks, the supernatant is collected by centrifugation at 10000rpm for 10min at 4 ℃, and the serum antibody titer is detected by an indirect ELISA method. As shown in figures 3-6, the IgM expression of group C frog is obviously up-regulated at different time points compared with other groups, the antibody titer is always the highest, the AD group does not detect the antibody, the IgM expression level is also high, and the BE treatment group is at an intermediate level, which indicates that the aloe vector inactivated vaccine soaking treatment mode is the most effective.
Table 1A list of primers used
Embodiment III
On the basis of the first and second embodiments, the following description is given of the effect of different vaccines on Ictalurus punctatus infected with Yersinia ruckeri, unlike the second embodiment, on the aquatic subjects involved in Ictalurus punctatus
The yersinia ruckeri related to the invention is preserved by the fishing medicine laboratory of the aquatic product research institute of the national institute of aquatic science. Mashing aloe (prepared from purchased plant), dissolving, removing residues, and keeping supernatant at 4deg.C.
Yersinia ruckeri is inoculated in a BHI culture medium, shake-cultured for 18 hours at 25-28 ℃, preferably 28 ℃, and inactivated whole bacteria vaccine is prepared by inactivating bacterial liquid with 0.5% formaldehyde, and the presence or absence of viable bacteria is detected by using a flat plate experiment to evaluate the inactivation effect. Mixing the inactivated vaccine with aloe supernatant at a ratio of 1:1, and diluting to 20%. The channel catfish is randomly grouped, 30 channel catfish are respectively provided with an aloe soaking group (A), an aloe carrier inactivated vaccine injection group (B), an aloe carrier inactivated vaccine soaking group (C), an inactivated vaccine soaking group (D) and an inactivated vaccine injection group (E), and the grouping mode is a treatment mode. Infection experiments are carried out on the channel catfish after 2 weeks of vaccine injection by injecting Yersinia ruckeri into the abdominal cavity, and the state and the morbidity and mortality of the channel catfish are observed and recorded. . The test was performed in a 100cm by 50cm by 60cm aquarium with water changed daily and maintained at a water temperature of about 26.+ -.1 ℃ using approximately 200g channel catfish in 100 feet per group and three replicates per group. The research results are shown in fig. 2, the survival number of the channel catfish in the group C is highest, all channel catfish in the group A and the group D die, and the channel catfish in the group BE partially survives, so that the aloe carrier inactivated vaccine soaking treatment mode is most effective.
The channel catfish is randomly grouped, 30 channel catfish are respectively provided with an aloe soaking group (A), an aloe carrier inactivated vaccine injection group (B), an aloe carrier inactivated vaccine soaking group (C), an inactivated vaccine soaking group (D) and an inactivated vaccine injection group (E), and the grouping mode is a treatment mode. . The test was performed in a 100cm by 50cm by 60cm aquarium with water changed daily and maintained at a water temperature of about 26.+ -.1 ℃ using approximately 200g channel catfish in 100 feet per group and three replicates per group. Taking various channel catfish for blood drawing, extracting total RNA, reversing the total RNA into cDNA as qPCR template, and detecting lgM gene expression condition, wherein the primers are shown in table 1; the channel catfish blood of each group was collected at 1, 2, 4 and 8 weeks, and the supernatant was collected by centrifugation at 10000rpm for 10min at 4℃and serum antibody titers were detected by an indirect ELISA method. As shown in fig. 4-6, the IgM expression of the channel catfish in group C was significantly up-regulated at different time points than that in other groups, the antibody titer was always the highest, but no antibodies were detected in group AD, the IgM expression level was also high, and the BE treatment group was at intermediate level, indicating that the aloe vector inactivated vaccine soaking treatment mode was the most effective.
Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.
Claims (10)
1. A preparation method based on aloe and vaccine as carriers is characterized in that: the method comprises the following steps: firstly, inoculating bacteria into a culture medium, and shake culturing; secondly, inactivating the bacterial liquid to prepare an inactivated bacterial vaccine, and detecting whether viable bacteria exist or not to evaluate the inactivation effect; finally, the inactivated vaccine is mixed with aloe and diluted to form the carrier based on aloe and vaccine.
2. A method as claimed in claim 1, wherein: the bacteria are Elizabeth milbezoar, the culture medium is BHI, shake culture is carried out at 25-28 ℃ for 10-18 hours, the inactivated bacteria are whole inactivated bacteria, the detection is carried out by using a flat plate experiment, the mixing is carried out by mixing with aloe supernatant in a ratio of 1:1, and the dilution is carried out to 20% concentration.
3. The method of any one of claims 1-2, wherein formaldehyde is used by the fire fighting bacteria.
4. A method for applying a vector based on aloe and vaccine in aquatic animals is characterized in that: the first step: randomly grouping aquatic animals, and respectively arranging an aloe soaking group, an aloe vector inactivated vaccine injection group, an aloe vector inactivated vaccine soaking group, an inactivated vaccine soaking group and an inactivated vaccine injection group; and a second step of: soaking or injecting according to groups; and a third step of: detecting gene expression and detecting antibody titer.
5. The method of claim 4, wherein: after injection according to groups, bacterial infection experiments are also required to be carried out after the injection, and the state or morbidity and mortality of aquatic animals are observed.
6. The method of claim 4, wherein: and the detection gene expression is that blood of the aquatic animal is taken on the X-th day, total RNA is extracted, and the total RNA is inverted into cDNA as a qPCR template, so that lgM gene expression condition is detected.
7. The method of claim 4, wherein: and taking blood of each group of aquatic animals at the X week, centrifuging to collect supernatant, and detecting serum antibodies according to an indirect ELISA method.
8. The method of any one of claims 4-7, characterized by: the aquatic animal is bullfrog or channel catfish.
9. The method of claim 5, wherein: after the injection is performed according to the groups, the infection experiment is performed by injecting yersinia ruckeri into the abdominal cavity of the channel catfish, and the state and the morbidity and mortality of the channel catfish are observed and recorded.
10. A vector based on aloe and a vaccine, characterized in that: including aloe vera or vaccines.
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| CN202311234930.9A CN117159695A (en) | 2023-09-21 | 2023-09-21 | Vaccine based on aloe as carrier and application of vaccine in aquatic animals |
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| CN202311234930.9A CN117159695A (en) | 2023-09-21 | 2023-09-21 | Vaccine based on aloe as carrier and application of vaccine in aquatic animals |
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