CN102718866A - Method for preparing takifugu obscurus source resistant pathogenic aeromonas veronii egg-yolk antibody - Google Patents
Method for preparing takifugu obscurus source resistant pathogenic aeromonas veronii egg-yolk antibody Download PDFInfo
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Abstract
The invention discloses a method for preparing a takifugu obscurus source resistant pathogenic aeromonas veronii egg-yolk antibody, which comprises the steps of bacterin preparation, immunization, egg-yolk solution pretreatment, egg-yolk powder preparation and the like. The method disclosed by the invention has the advantages that the operation is easy, the effect is good, and through assay of the titer of immunized eggs collected every seven days, the titer assayed through ELISA (enzyme-linked immunosorbent assay) after the fourth immunization can reach 1:12560, and the titer of the egg-yolk antibody powder prepared further is constant.
Description
Technical field
The present invention relates to the technology of preparing of the pathogenic Aeromonas veronii yolk antibody in a kind of anti-fugu obscurus source, be used for the fish disease control that Aeromonas veronii causes.Belong to the biological products preparing technical field.
Background technology
Aeromonas veronii (Aeromonas veronii) claim all grand Aeromonass, Wei Luona Aeromonas again, nineteen eighty-three, the in honor of French microbiologist Veron of U.S.'s disease prevention and control center in the research of vibrios and Aeromonas contribution and name.This bacterium is prevalent in fresh water, sewage, and in soil and even the seawater, wherein a part of bacterial strain is a normal presence in the micro-ecological environment; And that other bacterial strains have is pathogenic, mainly infects poikilothermal animal, like mitten crab, loach, bright and beautiful carp; Spot fork-tail silver xenocypris etc. can cause mass mortality; Aeromonas veronii is a kind of opportunistic pathogen to warm blooded animal, but has the people to think that the immunologic function person of perfecting also can be infected, and acute diarrhea takes place the patient usually, also possibly cause microbemia, meningitis and endocarditis etc.In recent years, increasing case shows that Aeromonas veronii has become a kind of important people one fish and suffered from pathogenic bacterium altogether, and on food safety, shows significance, and part country has been defined as it Quarantine Objects of food safety.The use of antibacterials is important measures of animal production and epidemic prevention and control, but the use of the not science of antimicrobial drug causes drug residue, environmental pollution and bacterial resistance to become animal doctor's public health problem of a World Focusing.Therefore, seek a kind of safe, the antimicrobial drug substitute becomes a problem that urgency is to be solved efficiently.Special yolk antibody can be treated the disease that is caused by bacterium or virus infection, and have safety, noresidue, do not produce resistance, cheapness, advantage such as be easy to get, be a kind of microbiotic substitute with wide application prospect.
At present, yolk antibody development technology is just becoming the new focus of domestic and international field of medicaments.Yolk antibody concentration ability long term maintenance in the yolk is at the level (Larsson etc. that are equivalent to even surpass the blood AC; 1993), (Hatta etc., 1993); Every month laying period of the high egg fowl of one plumage, approximately can be produced 1500mg yolk antibody (Schade etc., 1994).The chemical nature of yolk antibody is a protein, can decompose be utilized as nutritive substance by body after playing a role, and pure article can residual any objectionable impurities; And stability better, and ability acidproof, alkaline-resisting, heat-resisting, anti-enzymolysis (Shimizu etc., 1992) can be made into the yolk antibody powder that makes things convenient for storage and transport, is a kind of ideal green biological products.The cardinal principle situation that present IgY technology is studied at home and used is: on veterinary science and development of functional food, relatively paid attention to, even more many western countries pay attention to product development and commercialization more.But the basic research work of IgY technical application is also relatively weaker, and almost nobody relates to its dosage form research, and this also becomes " bottleneck " of IgY widespread usage.
The research of IgY in the anti-smelting of disease is the focus and very active problem in passive immunization field.Aspect bacillary intestinal tract disease prevents and treats, think that at present IgY has three big effect mechanism: the one, the IgY of anti-specific pathogen bacterium can directly adhere on the cell walls of pathogenic bacteria, changes the pathogenic bacteria cell integrity, directly suppresses growth of pathogenic bacteria; The 2nd, IgY can adhere on the bacterial pilli, makes pathogenic bacteria can not adhere to intestinal mucosa; The 3rd, part IgY under the effect of enteron aisle digestive ferment, be degraded to little peptide (contain the little peptide that antibody is not held alterable height: the Fab part), these little peptides still can combine with antigen, in having with the cause of disease ability.Aspect the parenteral route diseases prevention and treatment; After IgY got into gi tract, under the effect of digestive ferment, IgY partly was degraded to little peptide; Very easily by intestinal absorption; Can combine with the adhesion factor of specific pathogen bacterium after getting into blood, make pathogenic bacteria not adhere to permissive cell and lose pathogenicly, can bring into play bacteriostatic action non-enteric pathogenic bacteria.Anti-cholecystokinin IgY can absorb with complete molecular form or is partially absorbed into blood in SD rat preduodenal section, bring into play its immunology and biological effect, but the mechanism of absorption of anti-CCK yolk antibody does not obtain further experimental demonstration.
Yolk antibody more existing reports of application aspect the anti-smelting of fish bacterial disease, Arastech etc. carry out the passive immunization prophylactic tria with yolk antibody to rainbow trout, and the discovery rainbow trout is taken yolk antibody and after 15 days Vibrio anguillarum has been produced tangible immunizing power.Gutierrez etc. carry out oral immunity with yolk antibody to Japanese eel, find that the quantity of Wdwardsiella tarda in its enteron aisle of immunity back significantly reduces, thereby suppress this cause of disease from injured intestines wall invasion liver and kidney, reduced should disease mortality ratio.Wang Bin etc. are adopting oral medication that turbot is carried out immunoprotection test discovery, no matter are to carry out the artificial challenge with intramuscular injection or with scratching the method for soaking, and can not make the fish morbidity, and the protection effect is better; Yolk antibody is better than the infectable infection group to scratching the protection effect of soaking infection.But wherein reason does not obtain further experimental demonstration yet.Usefulness special yolk antibody additives such as Li Xiaoliang show the immunoprotection experiment of Trionyx sinensis (Wiegmann): the fodder additives of specific IgY antibody has prophylactic effect preferably to corresponding cause of disease; Total plate count obviously reduces in the kidney, but IgY does not do clearer and more definite experimental demonstration in the intravital mechanism of action of Trionyx sinensis (Wiegmann) machine and metabolism.
Fugu obscurus (Takifugu fasciatus) is the special aquaculture object with high economic worth; Along with fugu obscurus artificial propagation, nutrition and nontoxic such as propagates artificially at the constantly perfect of integrated culture technology; The continuous expansion of fugu obscurus high-density, intensive culture scale, disease have become one of principal element of restriction the sector development.The fugu obscurus disease that Aeromonas veronii causes is existing the appearance in production practice, causes the certain economic loss.
Summary of the invention
The object of the present invention is to provide the preparation method of the pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source a kind of easy to operate, effective.
Technical solution of the present invention is:
The preparation method of the pathogenic Aeromonas veronii yolk antibody in a kind of anti-fugu obscurus source is characterized in that: comprise the following steps:
(1) with deposit number is the pathogenic Aeromonas veronii in fugu obscurus source (Aeromonas veronii) SY-R2 (depositary institution: Chinese typical culture collection center of CCTCC No:M2012154; Depositary institution address: Chinese Wuhan University; Preservation date: on May 3rd, 2012; Classification name: streak inoculation soybean casein agar culture medium flat plate Aeromonas veronii SY-R2); Cultivate 24 h for 37 ℃, picking list bacterium colony is coated the TSA flat board of diameter 15 cm, cultivates 20 h in 37 ℃; Wash lawn with aseptic PBS, measuring and adjusting bacterial concentration is 2.5 * 10
10CFU/ mL; The qualified back of pure inspection is by 37 ℃ of deactivation 24 h of 0.5% (V/V) formalin solution, and the qualified back of steriling test supplies preparation antigen; Per 5 weight part inactivated bacterial liquid add aluminium hydroxide gel 1 weight part of sterilization, and the Thiomersalate that adding is equivalent to inactivated bacterial liquid and aluminium hydroxide gel gross weight 0.01% is anticorrosion, obtains vaccine;
(2) immune programme for children: 28 the week age Luo Man laying hen carry out the wing venous blood collection as control serum, open and collect egg postpartum as experiment contrast; Open the 3rd day postpartum, with the vaccine chest muscle multi-point injection immunity of step (1) preparation, dosage 0.5mL/ only; Carry out the immunity second time after the first immunisation 1 week, immunizing dose 1.0mL/ only; The immunity back is every at a distance from the 20d booster immunization for the second time, continuous immunity 2 times, and immunizing dose only is 1.0mL/; The 4th immunity back 40d carries out immunity again, and immunizing dose is 2.0mL/; Begin from first immunisation, back 7 days wing venous blood collection separation of serum of each immunity, and collect all eggs, the egg that every interval 7d after the immunity is collected carries out titration; With having or not of micro-agglutination rough determination immune chicken serum and antibody from yolk;
(3) pre-treatment of yolk liquid: collect immunity back egg the 4th time, with being dipped in sterilization, airing in 0.5% SANIZOL C after the clear water grooming totally, under aseptic condition, isolate yolk, add the saline water of 1.5 times of volumes, high-speed homogenization prepares emulsion;
(4) preparation of powdery yolk: will carry out spraying drying through the pretreated yolk liquid of step (3), and make powdery yolk, wherein the pathogenic Aeromonas veronii yolk antibody in the promptly anti-fugu obscurus of IgY source.
The egg that step (2) is collected every interval 7d after the immunity; Isolate yolk under the aseptic condition; 80g/L polyoxyethylene glycol-8 000 (PEG-8 000) single stage method purification yolk antibody, ELISA measures antibody titer, and the yolk antibody of the 4th immunity back ELISA mensuration is tired and is 1:12560.
Spray-dired EAT is 120 ℃-140 ℃ in the step (4), and air outlet temperature is 68 ℃-72 ℃.
Step (3) high speed homogenate rotating speed is 9500r/min, and the time is 10-20min.
Deposit number is that the pathogenic Aeromonas veronii in fugu obscurus source (Aeromonas veronii) SY-R2 of CCTCC No:M2012154 obtains through the selection of the following step: get have classical symptom, heart, the liver lesion of the fugu obscurus in dead back a hour carry out the bacterium separation and Culture; Used substratum is a soybean casein agar culture medium flat plate; Cultivate 24h for 30 ℃; Choosing dominant bacteria, on the TSA flat board, to carry out pure culture pure until bacterium colony, obtains pathogenic strains; Said classical symptom is: belly expands, and anus is red and swollen, the water sample ascites that body surface and pectoral fin, abdomeinal fin and anal fin are incomplete, base portion is congested, hemorrhage, when dissected has a large amount of yellowish colour band blood; The liver enlargement is khaki color, and hemorrhage patch not of uniform size is arranged; Gall-bladder expands; Enteron aisle does not have food, is full of spumescence mucus, and intestines mucosa is hemorrhage.
The present invention is easy to operate, and is effective, and the yolk antibody of the 4th immunity back ELISA mensuration is tired and is 1:12560., the yolk antibody powder of further processing is tired constant.Comparing unique distinction of the present invention with other technologies also is: at first, antigen comes from fugu obscurus, and the fugu obscurus disease that Aeromonas veronii causes has never seen report at home and abroad, is a kind of brand-new disease of fugu obscurus.The second, be that the yolk antibody of antigen prepd was not met report at home and abroad equally with the Aeromonas veronii.The 3rd, the pre-treatment of the yolk antibody liquid described in this invention makes yolk antibody form emulsion, and this did not meet report in the preparation process of yolk antibody powder.
Below in conjunction with embodiment the present invention is described further.
Embodiment
The preparation method of the pathogenic Aeromonas veronii yolk antibody in a kind of anti-fugu obscurus source comprises the following steps:
(1) with deposit number is the pathogenic Aeromonas veronii in fugu obscurus source (Aeromonas veronii) SY-R2 streak inoculation soybean casein agar substratum (the Tryptose Soya Agar of CCTCC No:M2012154; TSA) flat board; Cultivate 24h for 37 ℃; Picking list bacterium colony is coated the TSA flat board of diameter 15cm, cultivates 20h in 37 ℃; Wash lawn with aseptic PBS (0.01M, pH 7.2), measuring and adjusting bacterial concentration is 2.5 * 10
10CFU/mL; The qualified back of pure inspection is by 37 ℃ of deactivation 24h of 0.5% (V/V) formalin solution, and the qualified back of steriling test supplies preparation antigen; Per 5 weight part inactivated bacterial liquid add aluminium hydroxide gel 1 weight part of sterilization, and the Thiomersalate that adding is equivalent to inactivated bacterial liquid and aluminium hydroxide gel gross weight 0.01% is anticorrosion, obtains vaccine;
(2) immune programme for children: 28 the week age Luo Man laying hen carry out the wing venous blood collection as control serum, open and collect egg postpartum as experiment contrast; Open the 3rd day postpartum, with the vaccine chest muscle multi-point injection immunity of step (1) preparation, dosage 0.5mL/ only; Carry out the immunity second time after the first immunisation 1 week, immunizing dose 1.0mL/ only; The immunity back is every at a distance from the 20d booster immunization for the second time, continuous immunity 2 times, and immunizing dose only is 1.0mL/; The 4th immunity back 40d carries out immunity again, and immunizing dose is 2.0mL/; Begin from first immunisation, back 7 days wing venous blood collection separation of serum of each immunity, and collect all eggs, the egg that every interval 7d after the immunity is collected carries out titration; With having or not of micro-agglutination rough determination immune chicken serum and antibody from yolk;
(3) pre-treatment of yolk liquid: collect immunity back egg the 4th time, with being dipped in sterilization, airing in 0.5% SANIZOL C after the clear water grooming totally, under aseptic condition, isolate yolk, add the saline water of 1.5 times of volumes, high-speed homogenization prepares emulsion;
(4) preparation of powdery yolk: will carry out spraying drying through the pretreated yolk liquid of step (3), and make powdery yolk, wherein the pathogenic Aeromonas veronii yolk antibody in the promptly anti-fugu obscurus of IgY source.
The egg that step (2) is collected every interval 7d after the immunity; Isolate yolk under the aseptic condition; 80g/L polyoxyethylene glycol-8 000 (PEG-8 000) single stage method purification yolk antibody, ELISA measures antibody titer, and the yolk antibody of the 4th immunity back ELISA mensuration is tired and is 1:12560.Spray-dired EAT is 120 ℃-140 ℃ in the step (4), and air outlet temperature is 68 ℃-72 ℃.
Step (3) high speed homogenate rotating speed is 9500r/min, and the time is 10-20min.Deposit number is that the pathogenic Aeromonas veronii in fugu obscurus source (Aeromonas veronii) SY-R2 of CCTCC No:M2012154 obtains through the selection of the following step: get have classical symptom, heart, the liver lesion of the fugu obscurus in dead back a hour carry out the bacterium separation and Culture; Used substratum is a soybean casein agar culture medium flat plate; Cultivate 24h for 30 ℃; Choosing dominant bacteria, on the TSA flat board, to carry out pure culture pure until bacterium colony, obtains pathogenic strains; Said classical symptom is: belly expands, and anus is red and swollen, the water sample ascites that body surface and pectoral fin, abdomeinal fin and anal fin are incomplete, base portion is congested, hemorrhage, when dissected has a large amount of yellowish colour band blood; The liver enlargement is khaki color, and hemorrhage patch not of uniform size is arranged; Gall-bladder expands; Enteron aisle does not have food, is full of spumescence mucus, and intestines mucosa is hemorrhage.
Claims (5)
1. the preparation method of the pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source is characterized in that: comprise the following steps:
(1) with deposit number is the pathogenic Aeromonas veronii in fugu obscurus source (Aeromonas veronii) the SY-R2 streak inoculation soybean casein agar culture medium flat plate of CCTCC No:M2012154; Cultivate 24h for 37 ℃; Picking list bacterium colony is coated the TSA flat board of diameter 15cm, cultivates 20h in 37 ℃; Wash lawn with aseptic PBS, measuring and adjusting bacterial concentration is 2.5 * 10
10CFU/mL; The qualified back of pure inspection is by 37 ℃ of deactivation 24h of 0.5% formalin solution, and the qualified back of steriling test supplies preparation antigen; Per 5 weight part inactivated bacterial liquid add aluminium hydroxide gel 1 weight part of sterilization, and the Thiomersalate that adding is equivalent to inactivated bacterial liquid and aluminium hydroxide gel gross weight 0.01% is anticorrosion, obtains vaccine;
(2) immune programme for children: 28 the week age Luo Man laying hen carry out the wing venous blood collection as control serum, open and collect egg postpartum as experiment contrast; Open the 3rd day postpartum, with the vaccine chest muscle multi-point injection immunity of step (1) preparation, dosage 0.5mL/ only; Carry out the immunity second time after the first immunisation 1 week, immunizing dose 1.0mL/ only; The immunity back is every at a distance from the 20d booster immunization for the second time, continuous immunity 2 times, and immunizing dose only is 1.0mL/; The 4th immunity back 40d carries out immunity again, and immunizing dose is 2.0mL/; Begin from first immunisation, back 7 days wing venous blood collection separation of serum of each immunity, and collect all eggs, the egg that every interval 7d after the immunity is collected carries out titration; With having or not of micro-agglutination rough determination immune chicken serum and antibody from yolk;
(3) pre-treatment of yolk liquid: collect immunity back egg the 4th time, with being dipped in sterilization, airing in 0.5% SANIZOL C after the clear water grooming totally, under aseptic condition, isolate yolk, add the saline water of 1.5 times of volumes, high-speed homogenization prepares emulsion;
(4) preparation of powdery yolk: will carry out spraying drying through the pretreated yolk liquid of step (3), and make powdery yolk, wherein the pathogenic Aeromonas veronii yolk antibody in the promptly anti-fugu obscurus of IgY source.
2. the preparation method of the pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus according to claim 1 source; It is characterized in that: the egg that step (2) is collected every interval 7d after the immunity; Isolate yolk under the aseptic condition; 80g/L polyoxyethylene glycol-8000 single stage method purification yolk antibody, ELISA measures antibody titer, and the yolk antibody of the 4th immunity back ELISA mensuration is tired and is 1:12560.
3. the preparation method of the pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus according to claim 1 source is characterized in that: spray-dired EAT is 120 ℃-140 ℃ in the step (4), and air outlet temperature is 68 ℃-72 ℃.
4. according to the preparation method of claim 1, the pathogenic Aeromonas veronii yolk antibody in 2 or 3 described anti-fugu obscurus sources, it is characterized in that: step (3) high speed homogenate rotating speed is 9500r/min, and the time is 10-20min.
5. according to the preparation method of claim 1, the pathogenic Aeromonas veronii yolk antibody in 2 or 3 described anti-fugu obscurus sources; It is characterized in that: deposit number is that the pathogenic Aeromonas veronii in fugu obscurus source (Aeromonas veronii) SY-R2 of CCTCC No:M2012154 obtains through the selection of the following step: get have classical symptom, heart, the liver lesion of the fugu obscurus in dead back a hour carry out the bacterium separation and Culture; Used substratum is a soybean casein agar culture medium flat plate; Cultivate 24h for 30 ℃; Choosing dominant bacteria, on the TSA flat board, to carry out pure culture pure until bacterium colony, obtains pathogenic strains; Said classical symptom is: belly expands, and anus is red and swollen, the water sample ascites that body surface and pectoral fin, abdomeinal fin and anal fin are incomplete, base portion is congested, hemorrhage, when dissected has a large amount of yellowish colour band blood; The liver enlargement is khaki color, and hemorrhage patch not of uniform size is arranged; Gall-bladder expands; Enteron aisle does not have food, is full of spumescence mucus, and intestines mucosa is hemorrhage.
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