CN101773672A - Oral biological agent for preventing and curing prawn leukopathia - Google Patents
Oral biological agent for preventing and curing prawn leukopathia Download PDFInfo
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- CN101773672A CN101773672A CN 201010130589 CN201010130589A CN101773672A CN 101773672 A CN101773672 A CN 101773672A CN 201010130589 CN201010130589 CN 201010130589 CN 201010130589 A CN201010130589 A CN 201010130589A CN 101773672 A CN101773672 A CN 101773672A
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- prawn
- bacillus subtilis
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Abstract
The invention discloses an oral biological agent for preventing and curing prawn leukopathia. The agent has the following components in percentage by weight: 1-10% of reformed Bacillus subtilis expressing VP28, 5-50% of chitosan and balance of corn starch used as a carrier. The agent has safe use, and can effectively prevent and cure prawn leukopathia syndromes.
Description
Technical field
The present invention relates to a kind of oral biological preparation, particularly a kind of oral biological preparation of preventing and treating prawn white spot disease.
Background technology
Shrimp white spot syndrome virus (white spot syndrome virus, WSSV, prawn white spot disease) causes acute, the lethal infectious disease of cultured prawn in the world wide, cause the tremendous economic loss of shrimp culture industry, this situation is particularly serious in China, has become one of principal disease that threatens China's shrimp culture industry.Nineteen ninety-five, International Office of Epizootics (OIE), FAO (Food and Agriculture Organization of the United Nation) (FAO) and Asian-Pacific area aquaculture development network center (NACA) classify it as one of aquatic animal viral blight that needs report.Guttate morphea syndrome does not still have the effectively preventing measure so far, is to attempt one of technical barrier that solves by the tackling key problem emphasis in the world.
Traditional early prevention is primarily aimed at pathogen, shrimp body and environment three factors, from improve breeding environment, the immunostimulant of throwing something and feeding improves the prawn resistance, cut off possible aspects such as route of transmission sets about preventing WSSV to infect.Discovered in recent years, invertebrates have acquired immune system (Kurtz﹠amp equally; Franz, 2003), for the exploratory development work that the WSSV based on the immunity of prawn Memorability prevents and treats is laid a good foundation.Utilizing the virus envelope albumen design biological preparation of having identified is one of thinking of WSSV control.Witteveldt et al. (2004) has made up WSSV envelope protein VP19 and VP28 reorganization bacterium respectively in escherichia coli, by purification injection and mixing oral way immune health prawn, different time infects by the mode of soaking after immunity then, the death condition of back prawn is infected in observation, find that VP19, VP28 have certain immune effect and anti-WSSV endurance, the immune effect of VP28 is better.Produce WSSV envelope protein VP19 and VP28 with silkworm as bioreactor, peroral immunity animal model Procambrus clarkii (Procambarus clarkii), relative survival rate can reach 89.5% and 94.7% (Xu et al., 2006) respectively behind the counteracting toxic substances.Du et al. (2006) utilize insect baculovirus as carrier at BmN expressed in insect cells VP28 albumen, crayfish is carried out injecting immune protection effect can reach 92%.After envelope protein VP19, VP28 express in Pichia sp. with the direct oral immunity crayfish of engineering bacteria, its relative death rate also obtains reduction to a certain degree, simultaneously in production reality, has certain feasibility (Jha et al., 2007) with the engineering bacteria peroral immunity.Thus, the protection effect of WSSV envelope protein is more satisfactory, especially the recombinant biological agent best results that designs with VP28.
Yet under the large-scale cultivation water environment, recombiant protein protection effect is unsatisfactory, by oral route especially, and antigen causes the deficiency of protection dosage in reasons such as digestive tract degraded, absorbance are low.
In recent years, bacillus subtilis is used for the production of pharmaceutical protein, antigen protein gradually, its advantage is presented as: 1) high secretion capacity, make recombinant expressed pharmaceutical protein do not need through the purification in downstream with separate, also make excretory antigen protein effectively submission to host's immune system cause immunne response; 2) antigen protein is showed in the spore outer surface, can be used as the application of oral vaccine; 3) characteristic of heat-resisting, the acidproof and salt tolerant of spore can tolerate gastrointestinal tract environment and be more conducive to and preserve; 4) quick, simple and direct fermentation and production technology; 5) this is as probiotic bacteria, if can play the effect of adjuvant as vaccine carrier.
The preparation method of VP28 recombined bacillus subtilis is at Ling Lin Fu,
Wei Fen LiHua Hua Du, Wei Dai, Zi Rong Xu.Oral vaccination with envelope protein VP28 against white spot syndromevirus in Procambarus clarkii using Bacillus subtilis as delivery vehicles.LettAppl Microbiol.2008,46 (5): 581-586 (Fu Linglin, the structure of shrimp white spot syndrome virus envelope protein VP28 hay engineering bacteria and the preparation and the disease resistance research thereof of VP28 yolk antibody. Zhejiang University's thesis for the doctorate, 2008) be disclosed.
Chitosan also has related article to inform the effect that it has the control prawn white spot disease.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of safe in utilization and can effectively prevent and treat the oral biological preparation of white spot syndrome.
In order to solve the problems of the technologies described above, the invention provides a kind of oral biological preparation of preventing and treating prawn white spot disease, the component of said preparation and weight content are: 1～10% expresses recombined bacillus subtilis and 5～50% chitosans of VP28, and all the other are the corn starch as carrier.
Improvement as the oral biological preparation of control prawn white spot disease of the present invention: the bacterial content of expressing the recombined bacillus subtilis of VP28 is 10
9Individual/gram.
In the present invention, the recombined bacillus subtilis of expressing VP28 can be according to Ling Lin Fu,
Wei Fen LiHua HuaDu, Wei Dai, Zi Rong Xu.Oral vaccination with envelope protein VP28 against whitespot syndrome virus in Procambarus clarkii using Bacillus subtilis as deliveryvehicles.Lett Appl Microbiol.2008,46 (5): 581-586 (Fu Linglin, the structure of shrimp white spot syndrome virus envelope protein VP28 hay engineering bacteria and the preparation and the disease resistance research thereof of VP28 yolk antibody. Zhejiang University's thesis for the doctorate, 2008) be prepared; Also can be to applicant of the present invention--Zhejiang University asks for.Chitosan and corn starch all can obtain by commercial mode; Wherein chitosan can be selected the feed grade chitosan for use, and corn starch can be crossed the 80-200 mesh sieve.
The storage conditions of the oral biological preparation of control prawn white spot disease of the present invention is: room temperature, effect duration is 2 years.
During the actual use of the oral biological preparation of control prawn white spot disease of the present invention, add in the prawn feed according to 0.1%～1% the ratio that accounts for the prawn feed gross weight, can the feeding prawn behind the uniform mixing.
Oral biological preparation of the present invention has the following advantages:
1) easy to use, can be used as feed additive and in prawn feed, use;
2) safety good, have no side effect;
3) prevention effect is good, significantly improves prawn immunity and survival rate; Given full play to and expressed the recombined bacillus subtilis of VP28 and the collaborative preventive and therapeutic effect of chitosan;
4) production technology is simple, cost is low, easy storage.
The specific embodiment
The following examples help those skilled in the art more fully to understand the present invention, but do not limit the present invention in any way.
Following % is weight %.
Embodiment 1, a kind of oral biological preparation of preventing and treating prawn white spot disease, its recombined bacillus subtilis by 5% expression VP28,25% chitosan and 70% corn starch (crossing the 80-200 mesh sieve) are formed.The bacterial content of expressing the recombined bacillus subtilis of VP28 is 10
9Individual/gram.
Preparation method is: above-mentioned three components is simply mixed get final product.
Experiment 1, Penaeus vannamei are cultured in the cement pit of 25 ± 1 ℃ of temperature controls, and be divided into 4 at random and organize greatly, every group of 3 repetitions, each repeats 20, supports a week temporarily.Day feeding volume is 2% of prawn body weight.
In the prawn feed of routine, add oral biological preparation (embodiment 1 gained) the feeding Penaeus vannamei of the control prawn white spot disease that accounts for prawn feed gross weight 0.5%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; As test group.
In the prawn feed of routine, add recombined bacillus subtilis (its effective dose is with the consumption sum of recombined bacillus subtilis and the chitosan of the expression VP28 in the test group) the feeding Penaeus vannamei of the expression VP28 that accounts for prawn feed gross weight 0.15%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; Organize 1 as a comparison;
In the prawn feed of routine, add chitosan (its effective dose is with the consumption sum of recombined bacillus subtilis and the chitosan of the expression VP28 in the test group) the feeding Penaeus vannamei that accounts for prawn feed gross weight 0.15%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; Organize 2 as a comparison;
With the prawn feed feeding Penaeus vannamei of routine 33 days, as the blank group.
After 33 days, use shrimp white spot syndrome virus (WSSV) (WSSV of 100 times of 50% lethal dose) to soak counteracting toxic substances respectively test group, contrast groups 1, contrast groups 2 and blank group, observed 10 days continuously behind the counteracting toxic substances, write down dead shrimp number, and detect virus with PCR.The result is as follows:
Test group prawn survival rate is up to 96.7%, and contrast groups 1 prawn survival rate reaches 75.0%, and contrast groups 2 prawn survival rates reach 25.0%, and the prawn survival rate of blank group only is 16.7%.Adopt PCR to detect virus to the shrimp that survives, the result is: it is all negative that test group and contrast groups 1 PCR detect virus, and contrast groups 2 and blank group PCR detection virus are all positive.
Embodiment 2, a kind of oral biological preparation of preventing and treating prawn white spot disease, its recombined bacillus subtilis by 10% expression VP28,50% chitosan and 40% corn starch (crossing the 80-200 mesh sieve) are formed.The bacterial content of expressing the recombined bacillus subtilis of VP28 is 10
9Individual/gram.
Experiment 2,
In the prawn feed of routine, add oral biological preparation (embodiment 2 gained) the feeding Penaeus vannamei of the control prawn white spot disease that accounts for prawn feed gross weight 0.1%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; As test group.
In the prawn feed of routine, add recombined bacillus subtilis (its effective dose is with the consumption sum of recombined bacillus subtilis and the chitosan of the expression VP28 in the test group) the feeding Penaeus vannamei of the expression VP28 that accounts for prawn feed gross weight 0.06%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; Organize 1 as a comparison;
In the prawn feed of routine, add chitosan (its effective dose is with the consumption sum of recombined bacillus subtilis and the chitosan of the expression VP28 in the test group) the feeding Penaeus vannamei that accounts for prawn feed gross weight 0.06%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; Organize 2 as a comparison;
With the prawn feed feeding Penaeus vannamei of routine 33 days, as the blank group.
All the other contents are with experiment 1.The result is as follows:
Test group prawn survival rate is up to 95.0%, and contrast groups 1 prawn survival rate is 70.0%, and contrast groups 2 prawn survival rates are 18.3%, and the prawn survival rate of blank group only is 15.0%.Adopt PCR to detect virus to the shrimp that survives, the result is: it is all negative that the PCR of test group and contrast groups 1 detects virus, and contrast groups 2 and blank group PCR detection virus are all positive.
Embodiment 3, a kind of oral biological preparation of preventing and treating prawn white spot disease, its recombined bacillus subtilis by 1% expression VP28,5% chitosan and 94% corn starch (crossing the 80-200 mesh sieve) are formed.The bacterial content of expressing the recombined bacillus subtilis of VP28 is 10
9Individual/gram.
Experiment 3,
In the prawn feed of routine, add oral biological preparation (embodiment 3 gained) the feeding Penaeus vannamei of the control prawn white spot disease that accounts for prawn feed gross weight 1%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; As test group.
In the prawn feed of routine, add recombined bacillus subtilis (its effective dose is with the consumption sum of recombined bacillus subtilis and the chitosan of the expression VP28 in the test group) the feeding Penaeus vannamei of the expression VP28 that accounts for prawn feed gross weight 0.06%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; Organize 1 as a comparison;
In the prawn feed of routine, add chitosan (its effective dose is with the consumption sum of recombined bacillus subtilis and the chitosan of the expression VP28 in the test group) the feeding Penaeus vannamei that accounts for prawn feed gross weight 0.06%, continuous flow rate 30 days, the conventional prawn feed feeding of reuse 3 days; Organize 2 as a comparison;
With the prawn feed feeding Penaeus vannamei of routine 33 days, as the blank group.
All the other contents are with experiment 1.The result is as follows:
Test group prawn survival rate is up to 93.3%, and contrast groups 1 prawn survival rate is 71.7%, and contrast groups 2 prawn survival rates are 16.7%, and the prawn survival rate of matched group only is 13.3%.Adopt PCR to detect virus to the shrimp that survives, the result is: it is all negative that test group and contrast groups 1 PCR detect virus, and contrast groups 2 and blank group PCR detection virus are all positive.
Above presentation of results, oral biological preparation of the present invention are effective, the safe in utilization and biological preparation easily of a kind of anti-prawn white spot disease.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (2)
1. oral biological preparation of preventing and treating prawn white spot disease is characterized in that the component of said preparation and weight content are: 1～10% expresses recombined bacillus subtilis and 5～50% chitosans of VP28, and all the other are the corn starch as carrier.
2. the oral biological preparation of control prawn white spot disease according to claim 1 is characterized in that: the bacterial content of the recombined bacillus subtilis of described expression VP28 is 10
9Individual/gram.
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| CN2010101305899A CN101773672B (en) | 2010-03-23 | 2010-03-23 | Oral biological agent for preventing and curing prawn leukopathia |
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| CN2010101305899A CN101773672B (en) | 2010-03-23 | 2010-03-23 | Oral biological agent for preventing and curing prawn leukopathia |
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| CN101773672B CN101773672B (en) | 2012-11-21 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102764433A (en) * | 2012-06-07 | 2012-11-07 | 无锡海健生物科技有限公司 | Prawn white spot syndrome virus multivalent vector vaccine and application thereof |
| CN103734511A (en) * | 2013-12-17 | 2014-04-23 | 浙江大学 | Application of tryptophol serving as white spot syndrome-resistant feed additive |
| CN112375708A (en) * | 2020-11-16 | 2021-02-19 | 上海海洋大学 | Preparation for resisting white spot syndrome virus and decapod iridovirus 1 and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1620892A (en) * | 2004-12-10 | 2005-06-01 | 中国海洋大学 | Compound microecological preparation for prawn |
| CN1899299A (en) * | 2006-07-03 | 2007-01-24 | 房如森 | Chitosan powder medicine for fishing and its producing process |
-
2010
- 2010-03-23 CN CN2010101305899A patent/CN101773672B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1620892A (en) * | 2004-12-10 | 2005-06-01 | 中国海洋大学 | Compound microecological preparation for prawn |
| CN1899299A (en) * | 2006-07-03 | 2007-01-24 | 房如森 | Chitosan powder medicine for fishing and its producing process |
Non-Patent Citations (2)
| Title |
|---|
| 《Fish & Shellfish Immunology》 20090110 S. Rajeshkumar等 Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus (WSSV) 429－437 1－2 第26卷, 第3期 2 * |
| 《Letters in Applied Microbiology》 20080328 L.L. Fu, W.F. Li, H.H. Du, W. Dai and Z.R. Xu Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkia using Bacillus subtilis as delivery vehicles 581－586 1－2 第48卷, 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102764433A (en) * | 2012-06-07 | 2012-11-07 | 无锡海健生物科技有限公司 | Prawn white spot syndrome virus multivalent vector vaccine and application thereof |
| CN102764433B (en) * | 2012-06-07 | 2014-02-05 | 无锡海健生物科技有限公司 | Prawn white spot syndrome virus multivalent vector vaccine and application thereof |
| CN103734511A (en) * | 2013-12-17 | 2014-04-23 | 浙江大学 | Application of tryptophol serving as white spot syndrome-resistant feed additive |
| CN103734511B (en) * | 2013-12-17 | 2015-04-29 | 浙江大学 | Application of tryptophol serving as white spot syndrome-resistant feed additive |
| CN112375708A (en) * | 2020-11-16 | 2021-02-19 | 上海海洋大学 | Preparation for resisting white spot syndrome virus and decapod iridovirus 1 and preparation method thereof |
| CN112375708B (en) * | 2020-11-16 | 2023-05-26 | 上海海洋大学 | Preparation for resisting white spot syndrome virus and Octopus iridovirus 1 and preparation method thereof |
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| CN101773672B (en) | 2012-11-21 |
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