CN103304664A - Method for preparing pathogenic aeromonas veronii egg yolk antibody - Google Patents
Method for preparing pathogenic aeromonas veronii egg yolk antibody Download PDFInfo
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Abstract
The invention discloses a method for preparing a pathogenic aeromonas veronii egg yolk antibody. The method comprises the following steps of: preparing a vaccine; carrying out an immunization procedure; pre-treating egg yolk liquid; preparing egg yolk powder and the like. The method for preparing the pathogenic aeromonas veronii egg yolk antibody is easy to operate, good in effect, and capable of carrying out titer measurement on eggs which are collected every 7 days after immunization; moreover, the titer measured by ELISA (Enzyme Linked Immunosorbent Assay) after the fourth immunization can reach 1:12560; furthermore the prepared egg yolk antibody powder is constant in titer.
Description
The application is application number: 201210181107.1, and the applying date: 2013.6.4, title " preparation method of the pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source " divides an application.
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 again all grand Aeromonass, Wei Luona Aeromonas, nineteen eighty-three, Center for Disease Control and Prevention in honor of French microbiologist Veron 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 normal presence in the micro-ecological environment, and that other bacterial strains have is pathogenic, and the main infection poikilothermal animal is such as mitten crab, loach, bright and beautiful carp, spot fork-tail silver xenocypris etc. can cause 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 occurs the patient usually, also may 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 altogether pathogenic bacterium, and shows significance in food safety, and Countries 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, efficient antimicrobial drug substitute and become a problem anxious to be resolved.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, the advantage such as be easy to get, be a kind of Substitutes For Antibiotic with wide application prospect.
At present, the yolk antibody Development Techniques is just becoming the new focus of domestic and international field of medicaments.Yolk antibody concentration energy long term maintenance in the yolk is at the level (Larsson etc. that are equivalent to even surpass the blood antibody concentration, 1993), (Hatta etc., 1993), the high egg fowl of one plumage laying period, approximately can be produced 1500mg yolk antibody (Schade etc., 1994) per month.The chemical nature of yolk antibody is protein, can decompose be utilized as nutritive substance by body after playing a role, and sterling can residual any objectionable impurities; And stability better, and energy 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 desirable green bio goods.The IgY technology is studied at home with the cardinal principle situation of using and is at present: relatively paid attention on veterinary science and development of functional food, even more many western countries pay attention to product development and commercialization more.But the basic research work that the IgY technology is used is also relatively weaker, and almost nobody relates to its dosage form research, and this also becomes " bottleneck " that IgY generally uses.
The research of lgY in the anti-smelting of disease is the focus in passive immunization field and very active problem.Aspect bacillary intestinal tract disease prevents and treats, think that at present IgY has three large effect mechanism: the one, the IgY of anti-specific pathogen bacterium can directly adhere on the cell walls of pathogenic bacteria, changes the integrity of pathogenic bacteria cell, directly suppresses the 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 is under the effect of enteron aisle digestive ferment, and (the little peptide that contains antibody end alterable height: the Fab part), these little peptides still can be combined with antigen, in having and the cause of disease ability to be degraded to little peptide.Aspect the parenteral route diseases prevention and treatment, after IgY enters gi tract, under the effect of digestive ferment, the IgY Partial digestion is little peptide, very easily by intestinal absorption, can be combined with the adhesion factor of specific pathogen bacterium after entering blood, make pathogenic bacteria not adhere to permissive cell and lose pathogenic, can be to non-enteric pathogenic bacteria performance bacteriostatic action.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, find that rainbow trout takes yolk antibody and after 15 days Vibrio anguillarum produced obvious immunizing power.Gutierrez etc. carry out oral immunity with yolk antibody to Japanese eel, find after the immunity that the quantity of Wdwardsiella tarda significantly reduces in its enteron aisle, 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.The 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 preferably prophylactic effect to corresponding cause of disease; total plate count obviously reduces in the kidney, but clearer and more definite experimental demonstration is not done in the mechanism of action and the metabolism of IgY in the Trionyx sinensis (Wiegmann) body.
Fugu obscurus (Takifugu fasciatus) is the special aquaculture object with high economic worth, along with fugu obscurus artificial propagation, nutrition and nontoxic the constantly perfect of integrated culture technology such as propagate artificially, 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 occurs in production practice, causes certain financial 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) the SY-R2(depositary institution of CCTCC No:M2012154: Chinese Typical Representative culture collection center, depositary institution address: Wuhan, China university, preservation date: on May 3rd, 2012, Classification And Nomenclature: Aeromonas veronii SY-R2) streak inoculation soybean casein agar culture medium flat plate, 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; Press 0.5%(V/V after pure inspection is qualified) 37 ℃ of deactivation 24h of formalin solution, steriling test is qualified rear for 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 in contrast serum of wing venous blood collection, 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; For the second time immunity is rear every the 20d booster immunization, continuous immunity 2 times, and immunizing dose only is 1.0mL/; 40d after the 4th immunity carries out immunity again, and immunizing dose is 2.0mL/; From first immunisation, rear 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 egg after the 4th immunity, be dipped in sterilization, airing in 0.5% bromogeramine after clean with the clear water grooming, isolate yolk under aseptic condition, add the physiological saline 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 i.e. pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source of IgY.
The egg that step (2) is collected every interval 7d after the immunity, isolate yolk under the aseptic condition, 80g/L PEG-8 000(PEG-8000) single stage method purification yolk antibody, ELISA measures antibody titer, and the yolk antibody that ELISA measures after the 4th immunity is tired and is 1:12560.
Spray-dired inlet temperature 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) the SY-R2 selection through the following steps of CCTCC No:M2012154 obtains: get have classical symptom, heart, the liver lesion of the fugu obscurus in dead rear a hour carry out the bacterium separation and Culture, used medium is soybean casein agar culture medium flat plate, cultivate 24h for 30 ℃, choose dominant bacteria and carry out pure culture until bacterium colony is pure at the TSA flat board, obtain pathogenic strains; Described 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; Liver enlargement is khaki color, and hemorrhage patch not of uniform size is arranged; Gall-bladder expands; Enteron aisle is full of spumescence mucus without food, and intestinal mucosa is hemorrhage.
The present invention is easy to operate, and is effective, and the yolk antibody that ELISA measures after the 4th immunity is tired and is 1:12560., the yolk antibody powder of further making 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, the yolk antibody of preparation was not met report at home and abroad equally take Aeromonas veronii as antigen.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.
The invention will be further described below in conjunction with embodiment.
The pathogenic Aeromonas veronii in fugu obscurus source, deposit number are CCTCC No:M2012154; Classification And Nomenclature: glossy ganoderma, Latin name: Aeromonas veronii SY-R2, depositary institution: Chinese Typical Representative culture collection center, depositary institution address: Wuhan, China university, preservation date: on May 3rd, 2012.
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 ℃; Use aseptic PBS(0.01M, pH7.2) wash lawn, measuring and adjusting bacterial concentration is 2.5 * 10
10CFU/mL; Press 0.5%(V/V after pure inspection is qualified) 37 ℃ of deactivation 24h of formalin solution, steriling test is qualified rear for 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 in contrast serum of wing venous blood collection, 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; For the second time immunity is rear every the 20d booster immunization, continuous immunity 2 times, and immunizing dose only is 1.0mL/; 40d after the 4th immunity carries out immunity again, and immunizing dose is 2.0mL/; From first immunisation, rear 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 egg after the 4th immunity, be dipped in sterilization, airing in 0.5% bromogeramine after clean with the clear water grooming, isolate yolk under aseptic condition, add the physiological saline 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 i.e. pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source of IgY.
The egg that step (2) is collected every interval 7d after the immunity, isolate yolk under the aseptic condition, 80g/L PEG-8 000(PEG-8000) single stage method purification yolk antibody, ELISA measures antibody titer, and the yolk antibody that ELISA measures after the 4th immunity is tired and is 1:12560.Spray-dired inlet temperature 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) the SY-R2 selection through the following steps of CCTCC No:M2012154 obtains: get have classical symptom, heart, the liver lesion of the fugu obscurus in dead rear a hour carry out the bacterium separation and Culture, used medium is soybean casein agar culture medium flat plate, cultivate 24h for 30 ℃, choose dominant bacteria and carry out pure culture until bacterium colony is pure at the TSA flat board, obtain pathogenic strains; Described 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; Liver enlargement is khaki color, and hemorrhage patch not of uniform size is arranged; Gall-bladder expands; Enteron aisle is full of spumescence mucus without food, and intestinal mucosa is hemorrhage.
Claims (1)
1. a method for preparing pathogenic Aeromonas veronii yolk antibody 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; By 37 ℃ of deactivation 24h of 0.5% formalin solution, steriling test was qualified rear for preparation antigen after pure inspection was qualified; 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 in contrast serum of wing venous blood collection, 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; For the second time immunity is rear every the 20d booster immunization, continuous immunity 2 times, and immunizing dose only is 1.0mL/; 40d after the 4th immunity carries out immunity again, and immunizing dose is 2.0mL/; From first immunisation, rear 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 egg after the 4th immunity, be dipped in sterilization, airing in 0.5% bromogeramine after clean with the clear water grooming, isolate yolk under aseptic condition, add the physiological saline 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 i.e. pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source of IgY;
Step (2) is isolated yolk to the egg that every interval 7d after the immunity collects under the aseptic condition, 80g/L PEG-8 000 single stage method purification yolk antibody, and ELISA measures antibody titer, and the yolk antibody that ELISA measures after the 4th immunity is tired and is 1:12560;
Spray-dired inlet temperature is 120 ℃-140 ℃ in the step (4), and air outlet temperature is 68 ℃-72 ℃.
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