CN101074260A - Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal - Google Patents
Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal Download PDFInfo
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Abstract
Production of various Aeromonas hydrophila protective antigen monovalent and multivalent vitelline antibody and its usage in prevention of aquatic animal diseases are disclosed. It utilizes minor-protective antibody to make immune irritant composite egged hen and produce vitelline antibody product. It's safe, efficient, fast, has no harm and residue. It has more output, better immune-system development and functions of nutrients-added, disease-prevention and health-care.
Description
Technical field
The present invention relates to a kind of biological immune technology, the preparation method of particularly a kind of each main protection antigen unit price of Aeromonas hydrophila that is applicable to aquatic animal passive immunization prevention and treatment, multivalent vitelline antibody, and their application in the aquatic animal disease control.
Technical background
Yolk antibody (IgY) has been widely used in preventing and treat bacterium or the virus disease of birds such as domestic animals such as pig, ox and chicken, duck, goose as new class medicine and feed supplement.But studies have shown that the preservation of purification yolk antibody and egg yolk liquid freeze-drying preservation or spraying drying, antibody activity is not suffered a loss, and acidproof, heat-resisting and stable performance.Multivalent vitelline antibody has no side effect, and life-time service can not cause pathogenic bacteria to produce resistance, be a kind of application prospect widely, the immunoglobulin (Ig) of stable in properties.Because yolk antibody has tolerance stomach en-and tryptic digestion within a certain period of time, therefore can pass through per os passive immunization approach, bring into play its immunologic competence or biologic activity (Hatta H) effectively.Most of IgY is by directly acting on pathogenetic bacteria in oral cavity or gi tract, stoping bacterium that sticking, invading of intestinal cell brought into play disease-resistant, health-care effect.
A key distinction that is different from terrestrial animal after aquatic animal disease is anti-is to be difficult to prevent and treat by the mode of injecting immune, and the above-mentioned characteristic of yolk antibody helps yolk antibody is applied to the control of aquatic products disease.Aeromonas hydrophila is the important pathogen of aquatic animal, and extracellular products and outer membrane protein are one of its main virulence factors, also is main protective antigen simultaneously.The yolk antibody of the main virulence factor of development Aeromonas hydrophila might suppress its breeding by the blocking-up Aeromonas hydrophila in the sticking, invades of enteron aisle, reaches the effect that the inhibition Aeromonas hydrophila is infected animal.But up to the present, preparation yolk antibody and the application of yolk antibody in the control of aquatic products disease still are in the starting stage, rare actual report.Chinese patent application: application number provides a kind of preparation technology of immune globulin against aeromonas hydrophila for the patent application of 200510060325.X, but the shortcoming of this technology is: 1) antigen that is adopted is full bacterium inactivation antigen, antigen particles is big, complicated component, antigen-specific is relatively poor, and specific antibody titres is lower; 2) adopt freund adjuvant (FCA) to be that as the shortcoming of immunological adjuvant 1. FCA oil can cause tissue injury by the sarolemma layer, local tubercle and the sterility granuloma of forming damaged meat; 2. FCA contains mycobacterium, and tuberculin test is positive; 3. FCA has carcinogenesis; 4. thickness is injected relatively effort; 5. the immunological adjuvant freund adjuvant is bigger than normal to pungency and the toxic side effect of animal, can cause the laying rate of laying hen obviously to descend, and does not also meet the food safety requirement of meat animals, so the use that is under an embargo clinically.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art part, and a kind of preparation method simple, with low cost, the stability and high efficiency valency, bland to animal, that security is good, each main protection antigen unit price yolk antibody of Aeromonas hydrophila that makes is provided.
Another object of the present invention is to provide a kind of preparation method who contains specifically at the multivalent vitelline antibody of various main protection antigen.
The present invention also aims to provide Aeromonas hydrophila main protection antigen unit price, the application of multivalent vitelline antibody in the aquatic animal disease control.
The invention provides the preparation method of the various main protection antigen unit price of a kind of Aeromonas hydrophila yolk antibody, it is made by following method: at first extract main protection antigen from Aeromonas hydrophila; Each main protection antigen of extracting is made protective antigen immunostimulating complex ISCOMs respectively; The multiple thing of the immunostimulation of each main protection antigen is carried out immunity to laying hen respectively; The egg through protective antigen immunostimulating complex of the same race immunity of learning from else's experience behind the immune certain hour is got its yolk, adds 1-9 times of physiological saline or phosphoric acid buffer (PBS) mixes in yolk, is the yolk antibody suspension.
Main protection antigen in the Aeromonas hydrophila is made the yolk antibody product; the immunostimulating complex immunity laying hen that only needs a spot of main protection antigen to make; can produce yolk antibody a large amount of, that height is tired; therefore following advantage is arranged: the one, to little, do not influence laying rate by the laying hen pungency of immunity.The 2nd, yolk antibody output height, cost is low, and the convenience of gathering, production efficiency are higher than serum antibody, are convenient to industrialization production.The 3rd, effective with yolk antibody blocking-up pathogenic micro-organism Aeromonas hydrophila to infecting of aquatic animal, and nuisanceless, noresidue.Yolk antibody can not influence the normal bacterium colony in the enteron aisle only at specific antigen.The 4th, yolk is the conventional nutrition added ingredients of animal-feed, gives yolk diseases prevention, nourishing function and be a kind of innovation in the feed formulation design.The 5th, the diseases prevention of yolk antibody, nourishing function can design applied range according to clinical demand.The 6th, compare (as vaccine) with the active immunity preparation, fast, there is not safety issue in the yolk antibody effect.The 7th, lower for immunity system growth evolution level, based on the aquatic animal of non-specific immunity, yolk antibody has special advantages.The 8th, yolk antibody is made stable in properties behind the dry powder, is easy to preserve.
Main protection antigen in the described Aeromonas hydrophila or be outer membrane protein (MOMP) or for extracellular products (ECPs) or for adhesin.
The preparation method of described extracellular products (ECPs) is: 28 ℃ of 200r/min shaking table 24h cultivate the recovery Aeromonas hydrophila, and the amount with 1% is inoculated in the TSB nutrient solution, and 200r/min cultivates about 36h for 28 ℃.4 ℃ of centrifugal 20min of 10000r/min get supernatant, 0.45 μ m membrane filtration, 70% saturated ammonium sulphate, overnight leaving standstill, centrifugal collection, precipitation is resuspended with 0.02mol/L Tris (PH7.5), 4 ℃, to the dialysis of 500mlTris damping fluid, replacing liquid was 1 time in per 6 hours, totally three times, the Bradford method is surveyed protein concentration, and-20 ℃ of preservations are standby.
The preparation method of described outer membrane protein (MOMP) is: inoculated bacteria is in the TSB substratum that contains 1% sodium-chlor, 30 ℃ of about 24h of 150r/min shaking table, 10 000r/min, 0.02mol/L Tris-HCL (pH7.5) washing 3 times, be resuspended in the above-mentioned damping fluid of about 10mL, the about altogether 6min of 200W ultrasonic disruption, 7000r/min4 ℃, 10min collects supernatant, it is 1.2% (W/V) that interpolation N-sarcosyl (N-lauroylsarcosine sodium salt) makes its final concentration, mixing, 4 ℃, overnight leaving standstill.4 ℃ of 40min of 40 000r/min, lower sediment is major outer membrane albumen, and the resuspended Bradford method of distilled water is measured protein concentration, packing ,-20 ℃ of preservations are standby.
The same outer membrane protein of described adhesin preparation method, or the Aeromonas hydrophila adhesin (method slightly) of expressing with genetic engineering technique.
One of method of each main protection antigen in the Aeromonas hydrophila being made epidemic disease stimulation composite I SCOMs is:
1. concentrate protective antigen to 2mg/ml;
2. handled 2 hours with the acidifying of 0.25M citric acid, make its pH drop to 2.5;
3. add Mega-10 to final concentration be 2%, 37 ℃, act on 4 hours;
4. add Quil A to final concentration be 0.05%～0.1%, add cholesterol (dissolving with chloroform in advance) then, Yelkin TTS to final concentration is 125ug/ml;
5. the ice-bath ultrasonic ripple is handled 8～10mins, and the negative pressure of vacuum dechlorination is imitative;
6. with 0.01M citric acid treatment dialysis 4h;
7. with 0.01M PBS (pH 7.2) dialysis 72 hours, changed liquid once in per 4 hours, the reaction solution through dialysing is protective antigen ISCOMs.-20 ℃ of preservations.
The authentication method of the immunostimulating complex that each protective antigen of Aeromonas hydrophila is made, be example with extracellular products, outer membrane protein and three kinds of immunostimulating complexs of making of adhesin:
The observation and the evaluation of extracellular products immunostimulating complex, outer membrane protein and adhesin immunostimulating complex: the sample 10ul that gets the three drips copper mesh, dye with 2% Tungstophosphoric acid, sodium salt, on the JEM-120EX Electronic Speculum, observe and identify, extracellular products immunostimulating complex, outer membrane protein immunostimulating complex and adhesin immunostimulating complex should be typical cage grating texture, and size is about the 40nm (see figure 1).
The immunostimulating complex that main protection antigen is made can be the concrete steps that laying hen carries out immunity respectively:
The method of laying hen being carried out immunity is: each protective antigen immunostimulating complex is carried out once laying hen or once above immunity; each immunizing dose is g/ chickens of 30 μ g-60 μ; being spaced apart about 15 days between twice immunity collected the last immunity egg that laying hen gave birth in 15-75 days later.
The best approach of described each protective antigen immunostimulating complex immunity laying hen is:
Employing is carried out the method that laying hen carries out three immunity: immunizing dose for the first time: g/ chicken of 30 μ g-60 μ, and it is immune that second time carried out in immunity for the first time in back 15 days, immunizing dose: g/ chicken of 40 μ g-80 μ; Immunity was for the third time carried out in immunity for the second time in back 15 days, immunizing dose: g/ chicken of 40 μ g-80 μ, the immunity back begins to collect in continuous 60 days egg after the 15th day for the third time.
The described way of extracting yolk from egg is: after egg shell is carried out disinfection, get yolk again and use.
With egg shell sterilization back, get yolk again and can prevent adherent bacterial contamination yolk product on the eggshell.
The egg shell method of disinfecting is had a lot, it or:
With getting yolk behind the iodine disinfection egg shell again or with getting yolk again behind the easy crin of 1ppm (10% double-stranded chlorination polyamine salt) or other surface disinfectants sterilization shell.
A kind of preparation method who contains the multivalent vitelline antibody of the various main protection antigen of Aeromonas hydrophila; be each main protection antigen ISCOMs of preparing by above-mentioned method, it is carried out immunity to laying hen respectively; get yolk respectively; in all yolk of being got, add 1-9 times of sterile saline or phosphoric acid buffer (PBS) is made suspension, two or more yolk antibody suspension equal proportion mixed solution is the main protection antigen multivalent vitelline antibody of Aeromonas hydrophila.
Be advance and the good result that shows preparation method of the present invention, the present invention has carried out titration and specific assay to the Aeromonas hydrophila main protection antigen yolk antibody that adopts present method preparation.
Above-mentioned prepared tiring of Aeromonas hydrophila main protection antigen yolk antibody is determined as follows:
1. the mensuration that above-mentioned prepared yolk antibody ELISA tires:
With extracellular products, outer membrane protein and the adhesin of Aeromonas hydrophila respectively by the amount coated elisa plate of 10 μ gmL-1 protein concns, every hole 50 μ L, 4 ℃ are spent the night.Dry solution in the hole, with the PBS-T solution room temperature sealing that contains 2%BSA 1 hour, the washing back added yolk antibody sample to be checked (making doubling dilution since 1: 20), every hole 50 μ L, room temperature reaction 1 hour, PBS-T washing 3 times, the goat-anti chicken HRP traget antibody that adds dilution in 1: 20000 then, every hole 50 μ L, room temperature reaction 1h, the same washing 3 times, with distilled water flushing 1 time, the substrate OPD that adds 50 μ L at last, color development at room temperature 15min uses the 2molL-1H2SO4 termination reaction.Read the OD value with enzyme connection instrument at the 495nm place.Antibody positive criterion is P/N 〉=2.1 (being positive hole/negative control hole ratio 〉=2.1).
2. adopt SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (Western-blot) to carry out specific assay:
Adopt discontinuous vertical gel electrophoresis, 3% concentrates glue, 12% separation gel.Protein adopts Coomassie brilliant blue R-250 dyeing to observe, analyze each protein component of the Aeromonas hydrophila of being extracted.
Immunoblotting: above-mentioned tropina is through SDS-PAGE) directly albumen is transferred on the nitrocellulose filter after the electrophoretic separation without Coomassie brilliant blue R-250 dyeing, get the nitrocellulose filter after the transfer printing, be positioned in the PBAT damping fluid of 3%BSA, room temperature is slowly vibrated and was sealed 1 hour, with PBAT washing 3 times, after each 3 minutes, add 1: 100 dilution yolk antibody (is anti-), room temperature was slowly vibrated 1 hour, with PBAT washing 3 times, each 3 minutes, add the goat-anti chicken serum IgG (two anti-) of the HRP enzyme labelling of dilution in 1: 20000 then, room temperature was slowly vibrated 1 hour, with PBAT washing 3 times, each 3 minutes,, add sedimentation type substrate DAB (Sigma) at last and develop the color with sterilized water washing 1 time.
Not preview protein standard M1 (97kD, 66kD, 43kD, 31kD, 20.1kD of gel electrophoresis, 14.4kD) (magnificent), preview protein standard M2 (175KD, 83kD, 62kD, 48kD are adopted in the trace test, 33kD, 25kD, 17kD, 7kD) (Niu Yinglun biotech company).
Aeromonas hydrophila main protection antigen unit price, the application of multivalent vitelline antibody in the aquatic animal disease control.
Be example with the experiment below, Aeromonas hydrophila main protection antigen unit price, the application of multivalent vitelline antibody on aquatic products diseases prevention and treatment animal are described.
The malicious protection test of attacking of feeding animal behind the multivalent vitelline antibody:
Mouse attack malicious protection test: test mice is a Kunming mouse, and available from laboratory animal plant of Medical University Of Fujian, specification: 20g/ is only.Attack malicious separating tests group and control group and carry out for two groups, every group of 13 mouse.Select with the tap water that contains 10% multivalent vitelline antibody mixed solution and drink to mouse test group every afternoon 3 select-6, connects to feed 12 days.Control group is drunk normal tap water.The 13rd day test group and control group mice are respectively got 2 blood samplings, and all the other every by abdominal injection 5 * 10
7The Aeromonas hydrophila of CFU (ZN1 strain) is observed 2 every day, continuous 7 days, calculates the mouse survival rate.The result shows that the Kunming mouse survival rate of throwing something and feeding through multivalent vitelline antibody is 72.7%, and the Kunming mouse survival rate of the yolk antibody of not throwing something and feeding only 27.3%.
Table 1: experimental group and control group mice are attacked the survival rate behind the poison
| Group | Confession examination mouse (only/group) | Drink the fate of antibody | Challenge dose (CPU/ only) | The 48h death toll | The 168h death toll | Survival rate (%) |
| Yolk antibody is | 11 | 12 | 5×10 7 | 3 | 3 | 72.7 |
| | 11 | 0 | 5×10 7 | 8 | 8 | 27.3 |
Common eel attack malicious protection test: the experiment European eel is available from permanently happy certain the common eel plant in Fujian Province, specification: 80g/ tail.Attack two groups of malicious separating tests group and control groups, every group 10 tail eel.Common eel is cultured in the 40L aquarium of laboratory, about temperature 22-25 ℃, changes water 1/3 every day.Respectively feeding in test group every morning and afternoon contains the black mole material of 10% multivalent vitelline antibody mixed solution, and a day feeding volume is the 0.8-1.0% of eel body weight, connects and feeds 15 days.Control group tap water spice just commonly used is thrown something and fed.Throw something and feed the 13rd day test group in back and control group respectively got 1 tail eel and gathered serum, and the every tail of remaining eel is by abdominal injection 1 * 10
8The Aeromonas hydrophila of CFU (ZN1 strain) is observed 2 every day, in continuous 2 weeks, calculates the eel survival rate.The result shows that the eel survival rate of throwing something and feeding through yolk antibody is 77.8%, and the common eel survival rate of the yolk antibody of not throwing something and feeding only 44.4%.Show that the resistance of throwing something and feeding yolk antibody the Europe eel of group attacking poison to Aeromonas hydrophila obviously strengthens.
Table 2: experimental group and control group common eel are attacked the survival rate behind the poison
| Group | For examination Europe eel (tail/group) | The antibody fate of throwing something and feeding | Attack toxic agent amount (CPU/ tail) | The dead mantissa of Europe eel in 2 weeks | Survival rate % |
| The yolk antibody group of throwing something and feeding | 9 | 15 | 1×10 8 | 2 | 77.8 |
| Control group | 9 | 0 | 1×10 8 | 5 | 44.4 |
This shows, through the feed addition manner animal of throwing something and feeding, can give the dip-dye ability of mouse and common eel opposing Aeromonas hydrophila according to the outer membrane protein yolk antibody of present method preparation and extracellular products yolk antibody.
Below will be to being that yolk antibody that antigen is made is respectively tired and the disease-resistant principle of specificity analyses and multivalent vitelline antibody that both are mixed and made into is carried out careful elaboration with outer membrane protein and extracellular products.
When outer membrane protein (MOMP), extracellular products (ECPs) or adhesin antigen immune with Aeromonas hydrophila stimulate mixture immunity laying hen, rise after the immunity 15 days for the third time, surveyed once in per 7 days, detected yolk antibody ELISA in continuous 60 days and tire, the yolk antibody with the coli common pili protein Preparation compares simultaneously.
Elisa assay shows, prepared Aeromonas hydrophila MOMP, ECPs and coli common pili albumen yolk antibody in the 15-60 after immunity for the third time days all reaches more than 1: 10280 corresponding immunizing antigen reaction titre separately, sees Table 3.Wherein the yolk antibody of Aeromonas hydrophila main protection antigen and coli common pili albumen yolk antibody can react with Aeromonas hydrophila MOMP antigen; show that prepared Aeromonas hydrophila MOMP yolk antibody can discern the specific antigen composition of outer membrane protein, point out the MOMP of Aeromonas hydrophila on antigenic structure, to have similar antigenic determinant simultaneously with coli common pili albumen.
It is 1: 2560 that Aeromonas hydrophila ECPs yolk antibody is tired to the ELISA of ECPs, being lower than this yolk antibody tires to MOMP is antigenic, Aeromonas hydrophila MOMP yolk antibody and ECPs antigen have weak cross reaction, infer the composition that also has MOMP in the ECPs antigen thus, though and these MOMP composition content are few, antigenicity is stronger; Coli common pili yolk antibody and Aeromonas hydrophila ECPs antigen do not react, infer that there are not common antigenic determinant in ECPs antigen and coli common pili antigen, or common antigenic determinant content rareness, or the MOMP component that contains in the ECPs antigen seldom, sees Table 3.
With Aeromonas hydrophila MOMP, ECPs and adhesin yolk antibody in preparation with the dilution of physiological saline equivalent different be, the yolk antibody mixture is in preparation process, because 2 kinds of yolk antibodies are mixed earlier and after add the dilution of equivalent physiological saline again, therefore single ELISA that plants composition reduce by half (table 3) of tiring.This result illustrates that also two kinds of methods can both effectively prepare yolk antibody.Yolk antibody mixture preparation process is simple, has kept the yolk nutritive substance in constant tiring, and is suitable as the Animal nutrition additive.
Table 3:MOMP, ECP yolk antibody antibody titer are measured
| Envelope antigen | One is anti- | 3 exempt to tire in back 15 days |
| The MOMP of 3 strain bacterium | The | 1∶10240 |
| The | 1∶10240 | |
| The | 1∶5120 | |
| The coli common | 1∶10240 | |
| The ECPs of 3 strain bacterium | The | 1∶160 |
| The | 1∶2560 | |
| The | 1∶1280 | |
| The coli common pili yolk antibody | ＜1∶20 |
Immunoblotting (Western-blot) result shows, outer membrane protein (MOMP) yolk antibody to the outer membrane protein of the Aeromonas hydrophila of 7 strain different serotypes and adhesin specific band all can in conjunction with, illustrate that prepared yolk antibody all has the antigen antibody reaction characteristic (Fig. 2) of broad spectrum to the Aeromonas hydrophila of different serotypes.
Each main protection antigen immunostimulating complex of Aeromonas hydrophila of the preparation of the present invention simultaneously has following advantage:
The Aeromonas hydrophila outer membrane protein ISCOMs immunocomplex of this laboratory by the outer membrane protein (MOMP) of 4 kinds of Aeromonas hydrophila strains A CTT10501, L316, TPS30, WC-2 is made; respectively eel is carried out immunity; immunity was carried out Aeromonas hydrophila TPS30 to eel in back 40 days and is attacked poison; the result shows; the protection ratio of Aeromonas hydrophila being attacked poison through the eel of Aeromonas hydrophila MOMP-ISCOMs immunity reaches 80%-100% respectively; and non-immune eel protection ratio is 0 (Dong Chuanfu etc., 2005).
Aeromonas hydrophila extracellular products (ECPs) is also obtained analog result to the immunoprotection test of eel.The Aeromonas hydrophila ECPs-ISCOMs that Fang Qinmei etc. (2004) make the extracellular products (ECPs) of Aeromonas hydrophila bacterial strain TPS30 divides immune group and second immunisation group that eel is carried out immunity.In once immune back 45 days, or behind the second immunisation 15, immune fish is attacked poison with Aeromonas hydrophila TPS30; the result shows; once immunity back protection ratio is through reaching 87.5%, behind the second immunisation protection ratio through reaching 100%, and not immune eel to attack poison back protection ratio be 0.
But outer membrane protein, extracellular products or adhesin are directly made immunological reagent and are carried out immunity and have following shortcoming:
1) need to extract a large amount of extracellular productses, outer membrane protein, prepare immunostimulating complex technical requirements height in a large number, cost is big;
2) it is big aquatic animal to be carried out the injecting immune operation easier, poor practicability; Direct immunization produces the effect time need be more than two weeks.
Description of drawings
Fig. 1 is the electron microscopic observation figure of immunostimulating complex
Fig. 2 is the Western-blot coloration result of Aeromonas hydrophila outer membrane protein yolk antibody to the Aeromonas hydrophila MOMP of 7 kinds of different serotypes.
Embodiment
Embodiment one:
A kind of method for preparing Aeromonas hydrophila outer membrane protein protective antigen unit price yolk antibody; at first from described Aeromonas hydrophila, obtain protective antigen-outer membrane protein; described outer membrane protein is for to be made by following method: inoculated bacteria is in the TSB substratum that contains 1% sodium-chlor; 30 ℃ of about 24h of 150r/min shaking table; 10000r/min; 0.02mol/L Tris-HCL (pH7.5) washing 3 times; be resuspended in the above-mentioned damping fluid of about 10mL; the about altogether 6min of 200W ultrasonic disruption; 4 ℃ of 7000r/min; 10min collects supernatant; it is 1.2% (W/V) that interpolation N-sarcosyl (N-lauroylsarcosine sodium salt) makes its final concentration; mixing, 4 ℃, overnight leaving standstill.4 ℃ of 40min of 40 000r/min, lower sediment is major outer membrane albumen, and the resuspended Bradford method of distilled water is measured protein concentration, packing ,-20 ℃ of preservations are standby.Then the major outer membrane albumen that is obtained is made immunostimulating complex ISCOMs, the method for making immunostimulating complex is:
1. concentrate protective antigen to 2mg/ml;
2. handled 2 hours with the acidifying of 0.25M citric acid, make its pH drop to 2.5;
3. add Mega-10 to final concentration be 2%, 37 ℃ of cracking 4 hours;
4. add Quil A to final concentration be 0.05%～0.1%, add cholesterol (dissolving with chloroform in advance) then, Yelkin TTS to final concentration is 125ug/ml;
5. the ice-bath ultrasonic ripple is handled 8～10mins, and the negative pressure of vacuum dechlorination is imitative;
6. with 0.01M citric acid treatment dialysis 4h;
7. with 0.01M PBS (pH 7.2) dialysis 72 hours, changed liquid once in per 4 hours, the reaction solution through dialysing is outer membrane protein immunostimulating complex ISCOMs, with its freezing preservation.
Again the outer membrane protein immunostimulating complex is carried out immunity to laying hen; immunity is for adopting immunity once; get the egg yolk (being generally more than 15 days) of the laying hen behind the immune certain hour; the physiological saline that adds about 3 times in yolk mixes, and is the main protection antigen yolk antibody of Aeromonas hydrophila.The freezing preservation of described yolk antibody is standby.
It is same as the prior art that present embodiment is not stated part.
Embodiment two:
All the other steps of present embodiment are identical with embodiment one with method, just the major outer membrane protein immunization with being obtained wherein stimulates the method for mixture immunity laying hen to change three immunity into to carry out, its concrete grammar is: immunizing dose for the first time: g/ chicken of 30 μ g-60 μ, the immunity second time, immunizing dose: g/ chicken of 40 μ g-80 μ were carried out in immunity for the first time in back 15 days; Immunity was for the third time carried out in immunity for the second time in back 15 days, immunizing dose: g/ chicken of 40 μ g-80 μ, the immunity back begins to collect in continuous 60 days egg after the 15th day for the third time.And the method for extracting yolk from egg is with getting yolk behind the iodine disinfection egg shell again.
Embodiment three:
All the other steps of present embodiment are identical with embodiment two, just wherein extract the method for yolk and get yolk again after adopting 10% double-stranded chlorination polyamine salt to the egg shell sterilization.
Embodiment four:
The step of present embodiment is identical with embodiment one with method, just changes physiological saline wherein into phosphoric acid buffer.
Embodiment five:
Method and the step of present embodiment and embodiment one are basic identical; just change outer membrane protein wherein into extracellular products; a kind of method for preparing Aeromonas hydrophila extracellular products protective antigen unit price yolk antibody; at first from Aeromonas hydrophila, obtain extracellular products; its method adopts conventional method to carry out; be specially 28 ℃ of 200r/min shaking table 24h and cultivate the recovery Aeromonas hydrophila, the amount with 1% is inoculated in the TSB nutrient solution, and 200r/min cultivates about 36h for 28 ℃.4 ℃ of centrifugal 20min of 10000r/min get supernatant, 0.45 μ m membrane filtration, 70% saturated ammonium sulphate, overnight leaving standstill, centrifugal collection, precipitation is resuspended with 0.02mol/LTris (PH7.5), 4 ℃ the Tris damping fluid fully dialysed, the Bradford method is surveyed protein concentration, and-20 ℃ of preservations are standby.The extracellular products that obtained is made Aeromonas hydrophila extracellular products yolk antibody according to the method for embodiment one.
It is identical with embodiment one that present embodiment is not stated part.
Embodiment six: method and the step of present embodiment and embodiment one are basic identical; just change outer membrane protein wherein into adhesin; a kind of method for preparing Aeromonas hydrophila adhesin protective antigen unit price yolk antibody makes Aeromonas hydrophila adhesin yolk antibody with the adhesin that obtained according to the method for embodiment one.
Embodiment seven:
A kind of preparation method who prepares each main protection antigen multivalent vitelline antibody of Aeromonas hydrophila; main protection antigen wherein is outer membrane protein, extracellular products and adhesin; after the three makes immunostimulating complex respectively by the method in the foregoing description earlier; laying hen is carried out immunity; after the egg that is obtained got yolk respectively; mix the physiological saline that adds 3 times again and mix, be Aeromonas hydrophila main protection antigen yolk antibody mixture.
Embodiment eight:
Each main protection antigen multivalent vitelline antibody of Aeromonas hydrophila that embodiment seven is obtained is applied to the control of eel and other fish.Each main protection antigen yolk antibody mixture of Aeromonas hydrophila of preparation is added into eel feed in 3% ratio, throws something and feeds after the mixing, 15 days is a course of treatment.Also can be used as eel feedstuff additive takes for a long time.Its using method can be used with reference to the method for common eel feedstuff additive.
Embodiment nine
Itself and embodiment eight are basic identical, just change each main protection antigen multivalent vitelline antibody of Aeromonas hydrophila wherein into monovalent yolk antibody.
Claims (11)
1. the preparation method of each main protection antigen unit price yolk antibody of Aeromonas hydrophila, it is made by following method: at first extract main protection antigen from Aeromonas hydrophila; Each main protection antigen of extracting is made protective antigen immunostimulating complex ISCOMs respectively; The multiple thing of the immunostimulation of each main protection antigen is carried out immunity to laying hen respectively; The egg through protective antigen immunostimulating complex of the same race immunity of learning from else's experience behind the immune certain hour is got its yolk, adds 1-9 times of physiological saline or phosphoric acid buffer (PBS) mixes in yolk, is the yolk antibody suspension.
2. the preparation method of each main protection antigen unit price yolk antibody of Aeromonas hydrophila according to claim 1 is characterized in that: the main protection antigen in the described Aeromonas hydrophila or for outer membrane protein or for extracellular products or for adhesin.
3. the preparation method of each main protection antigen unit price yolk antibody of Aeromonas hydrophila according to claim 2; it is characterized in that: each protective antigen immunostimulating complex is carried out once laying hen or once above immunity; each immunizing dose is g/ chickens of 30 μ g-60 μ; being spaced apart about 15 days between twice immunity collected the last immunity egg that laying hen gave birth in 15-75 days later.
4. the preparation method of each main protection antigen unit price yolk antibody of Aeromonas hydrophila according to claim 3, it is characterized in that: laying hen is carried out three times immunity, immunizing dose for the first time: g/ chicken of 30 μ g-60 μ, the immunity second time, immunizing dose: g/ chicken of 40 μ g-80 μ were carried out in immunity for the first time in back 15 days; Immunity was for the third time carried out in immunity for the second time in back 15 days, immunizing dose: g/ chicken of 40 μ g-80 μ, the immunity back begins to collect in continuous 60 days egg after the 15th day for the third time.
5. the preparation method of each main protection antigen unit price yolk antibody of Aeromonas hydrophila according to claim 4 is characterized in that: the method for extracting yolk from egg is: after egg shell is carried out disinfection, get yolk again and use.
6. the preparation method of each main protection antigen unit price yolk antibody of Aeromonas hydrophila according to claim 5 is characterized in that: with getting yolk behind the iodine disinfection egg shell again or with the easy crin of 1ppm.
7. preparation method who contains the multivalent vitelline antibody of each main protection antigen of Aeromonas hydrophila; be to prepare each main protection antigen ISCOMs, again it is carried out immunity to laying hen respectively by the described method of claim 1; get yolk respectively; in all yolk of being got, add 1-9 times of physiological saline or phosphoric acid buffer (PBS) is made suspension, two or more yolk antibody suspension equal proportion mixed solution is the main protection antigen multivalent vitelline antibody of Aeromonas hydrophila.
8. one kind as claim 1 or each main protection antigen unit price of 7 described Aeromonas hydrophilas or the application of multivalent vitelline antibody in the aquatic animal disease control.
One kind as claim 1 or each main protection antigen unit price of 7 described Aeromonas hydrophilas or multivalent vitelline antibody as the aquatic animal feed Application of Additives.
10. each main protection antigen unit price of Aeromonas hydrophila as claimed in claim 8 or the application of multivalent vitelline antibody in the common eel diseases prevention and treatment.
11. each main protection antigen unit price of Aeromonas hydrophila as claimed in claim 9 or multivalent vitelline antibody are as the eel feed Application of Additives.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100087221A CN101074260A (en) | 2007-03-20 | 2007-03-20 | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100087221A CN101074260A (en) | 2007-03-20 | 2007-03-20 | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal |
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| CN101074260A true CN101074260A (en) | 2007-11-21 |
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| CNA2007100087221A Pending CN101074260A (en) | 2007-03-20 | 2007-03-20 | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102718866A (en) * | 2012-06-04 | 2012-10-10 | 南通大学 | Method for preparing takifugu obscurus source resistant pathogenic aeromonas veronii egg-yolk antibody |
| CN103304664A (en) * | 2012-06-04 | 2013-09-18 | 南通大学 | Method for preparing pathogenic aeromonas veronii egg yolk antibody |
| CN108659116A (en) * | 2018-05-22 | 2018-10-16 | 淮海工学院 | A kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody |
| CN111426843A (en) * | 2020-03-13 | 2020-07-17 | 东莞市东阳光生物药研发有限公司 | Detection kit for pichia pastoris host protein residue and application thereof |
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2007
- 2007-03-20 CN CNA2007100087221A patent/CN101074260A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102718866A (en) * | 2012-06-04 | 2012-10-10 | 南通大学 | Method for preparing takifugu obscurus source resistant pathogenic aeromonas veronii egg-yolk antibody |
| CN103304664A (en) * | 2012-06-04 | 2013-09-18 | 南通大学 | Method for preparing pathogenic aeromonas veronii egg yolk antibody |
| CN103304664B (en) * | 2012-06-04 | 2014-10-29 | 南通大学 | Method for preparing pathogenic aeromonas veronii egg yolk antibody |
| CN108659116A (en) * | 2018-05-22 | 2018-10-16 | 淮海工学院 | A kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody |
| CN108659116B (en) * | 2018-05-22 | 2021-04-02 | 淮海工学院 | Immunogen synthesis method for preparing aquatic pathogenic bacterium aeromonas cross-type antibody |
| CN111426843A (en) * | 2020-03-13 | 2020-07-17 | 东莞市东阳光生物药研发有限公司 | Detection kit for pichia pastoris host protein residue and application thereof |
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