CN103304664B - Method for preparing pathogenic aeromonas veronii egg yolk antibody - Google Patents
Method for preparing pathogenic aeromonas veronii egg yolk antibody Download PDFInfo
- Publication number
- CN103304664B CN103304664B CN201310233318.XA CN201310233318A CN103304664B CN 103304664 B CN103304664 B CN 103304664B CN 201310233318 A CN201310233318 A CN 201310233318A CN 103304664 B CN103304664 B CN 103304664B
- Authority
- CN
- China
- Prior art keywords
- immunity
- yolk
- antibody
- aeromonas veronii
- yolk antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000002969 egg yolk Anatomy 0.000 title claims abstract description 63
- 241000607574 Aeromonas veronii Species 0.000 title claims abstract description 34
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 12
- 102000002322 Egg Proteins Human genes 0.000 title abstract 6
- 108010000912 Egg Proteins Proteins 0.000 title abstract 6
- 235000013345 egg yolk Nutrition 0.000 title abstract 6
- 230000003053 immunization Effects 0.000 claims abstract description 18
- 235000013601 eggs Nutrition 0.000 claims abstract description 17
- 238000002649 immunization Methods 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000002965 ELISA Methods 0.000 claims abstract description 9
- 229960005486 vaccine Drugs 0.000 claims abstract description 7
- 230000036039 immunity Effects 0.000 claims description 36
- 241000980706 Takifugu obscurus Species 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 210000004369 blood Anatomy 0.000 claims description 11
- 239000008280 blood Substances 0.000 claims description 11
- 210000002966 serum Anatomy 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 6
- SMYKVLBUSSNXMV-UHFFFAOYSA-K aluminum;trihydroxide;hydrate Chemical compound O.[OH-].[OH-].[OH-].[Al+3] SMYKVLBUSSNXMV-UHFFFAOYSA-K 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 5
- 244000068988 Glycine max Species 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000005018 casein Substances 0.000 claims description 5
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 5
- 235000021240 caseins Nutrition 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000000839 emulsion Substances 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000002203 pretreatment Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 241000287828 Gallus gallus Species 0.000 claims description 3
- 230000009849 deactivation Effects 0.000 claims description 3
- 230000003370 grooming effect Effects 0.000 claims description 3
- 238000000265 homogenisation Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 3
- 210000002976 pectoralis muscle Anatomy 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 3
- 238000004448 titration Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 5
- 239000000843 powder Substances 0.000 abstract description 5
- 238000005259 measurement Methods 0.000 abstract 1
- 201000010099 disease Diseases 0.000 description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 12
- 244000052616 bacterial pathogen Species 0.000 description 7
- 208000032843 Hemorrhage Diseases 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- 241000607534 Aeromonas Species 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 210000004347 intestinal mucosa Anatomy 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 2
- 206010019842 Hepatomegaly Diseases 0.000 description 2
- 241000277275 Oncorhynchus mykiss Species 0.000 description 2
- 241000736919 Pelodiscus sinensis Species 0.000 description 2
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 2
- 238000003723 Smelting Methods 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 210000000436 anus Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000001079 digestive effect Effects 0.000 description 2
- 210000000232 gallbladder Anatomy 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 210000000006 pectoral fin Anatomy 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 238000006748 scratching Methods 0.000 description 2
- 230000002393 scratching effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000252087 Anguilla japonica Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000252185 Cobitidae Species 0.000 description 1
- 241000252233 Cyprinus carpio Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000222336 Ganoderma Species 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 241000157468 Reinhardtius hippoglossoides Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 241000872315 Takifugu fasciatus Species 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 241000544286 Vibrio anguillarum Species 0.000 description 1
- 206010047400 Vibrio infections Diseases 0.000 description 1
- 241001222098 Xenocypris Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 201000009840 acute diarrhea Diseases 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229940107137 cholecystokinin Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 208000010824 fish disease Diseases 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 229940126701 oral medication Drugs 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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, the fish disease causing for Aeromonas veronii control.Belong to biological products preparing technical field.
Background technology
Aeromonas veronii (Aeromonas veronii) claim again all grand Aeromonass, Wei Luona Aeromonas, nineteen eighty-three, the in honor of contribution of French microbiologist Veron in the research of vibrios and Aeromonas of Center for Disease Control and Prevention and naming.This bacterium is prevalent in fresh water, sewage, and in soil and even seawater, wherein a part of bacterial strain is normal presence in micro-ecological environment, and that other bacterial strains have is pathogenic, main infection poikilothermal animal, 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 people to think that the immunologic function person of perfecting also can be infected, and acute diarrhea occurs patient conventionally, 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 in food safety, shows significance, and Countries has been stipulated the Quarantine Objects into 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 being caused by bacterium or virus infection, and there is 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, 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 yolk is being equivalent to even surpass the level (Larsson etc. of blood antibody concentration, 1993), (Hatta etc., 1993), the high egg fowl of one plumage laying period, monthly approximately can be produced 1500mg yolk antibody (Schade etc., 1994).The chemical nature of yolk antibody is protein, after playing a role, can as nutritive substance, decompose be utilized by body, and sterling can residual any objectionable impurities; And stability is better, energy acidproof, alkaline-resisting, heat-resisting, resistance to enzymolysis (Shimizu etc., 1992), can be made into the yolk antibody powder that facilitates storage and transport, is a kind of desirable green bio goods.IgY technology is studied at home with the cardinal principle situation of applying and is at present: in 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 technology application is also weaker, and almost nobody relates to its dosage form research, and this also becomes " bottleneck " that IgY generally applies.
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 the three large mechanisms of action: 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 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: Fab part), these little peptides still can be combined with antigen, in having and cause of disease ability to be degraded to little peptide.Aspect parenteral route diseases prevention and treatment, IgY enters after gi tract, under the effect of digestive ferment, IgY Partial digestion is little peptide, very easily by intestinal absorption, after entering blood, can be combined with the adhesion factor of specific pathogen bacterium, 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 or be partially absorbed into blood with complete molecular form 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.
There are reports in the application of yolk antibody aspect the anti-smelting of fish bacterial disease, and Arastech etc. carry out passive immunization prophylactic tria with yolk antibody to rainbow trout, finds that rainbow trout takes yolk antibody, after 15 days, Vibrio anguillarum produced to obvious immunizing power.Gutierrez etc. carry out oral immunity with yolk antibody to Japanese eel, find that the quantity of Wdwardsiella tarda in rear its enteron aisle of immunity significantly reduces, thereby suppress this cause of disease from injured intestines wall invasion liver and kidney, have reduced this sick mortality ratio.Wang Bin etc. are adopting oral medication to carry out immunoprotection test discovery to turbot, no matter are to carry out artificial challenge with intramuscular injection or with scratching the method for soaking, and can not make fish morbidity, and protection effect is better; Yolk antibody soaks infection protection effect to scratching is better than infectable infection group.But wherein reason does not obtain further experimental demonstration yet.The use special yolk antibody additives such as Li Xiaoliang show the immunoprotection experiment of Trionyx sinensis (Wiegmann): the fodder additives of specific IgY antibody has good prophylactic effect to corresponding cause of disease; in kidney, total plate count obviously reduces, but clearer and more definite experimental demonstration is not done in the mechanism of action and the metabolism of IgY in 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 has 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:
A preparation method for the pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source, is characterized in that: comprise the following steps:
(1) by deposit number, be 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, in 37 ℃, cultivates 20h; With aseptic PBS, wash lower lawn, measuring and adjust bacterial concentration is 2.5 * 10
10cFU/mL; After pure inspection is qualified, press 0.5%(V/V) 37 ℃ of deactivation 24h of formalin solution, antigen is prepared in the qualified rear confession of steriling test; Every 5 weight part inactivated bacterial liquid add aluminium hydroxide gel 1 weight part of sterilizing, add that to be equivalent to the Thiomersalate of inactivated bacterial liquid and aluminium hydroxide gel gross weight 0.01% anticorrosion, obtain vaccine;
(2) immune programme for children: 28 week age, Luo Man laying hen carried out wing venous blood collection serum in contrast, opened and collected egg postpartum as experiment contrast; Open the 3rd day postpartum, the vaccine chest muscle multi-point injection immunity of preparing by step (1), dosage 0.5mL/ is only; Within after first immunisation 1 week, carry out immunity for the second time, immunizing dose 1.0mL/ only; For the second time after immunity every 20d booster immunization, continuous immunity 2 times, immunizing dose is 1.0mL/ only; 40d after the 4th immunity, then carry out immunity, immunizing dose is 2.0mL/; From first immunisation, latter 7 days wing venous blood collection separation of serum of each immunity, and collect all eggs, the egg of collecting at interval of 7d after immunity is carried out to 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 clear water grooming, isolate yolk under aseptic condition, add the physiological saline of 1.5 times of volumes, high-speed homogenization is prepared emulsion;
(4) preparation of powdery yolk: will spray through the pretreated yolk liquid of step (3) dry, and make powdery yolk, wherein the i.e. pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source of IgY.
Step (2) is to the egg of collecting at interval of 7d after immunity, under aseptic condition, isolate yolk, 80g/L PEG-8 000(PEG-8000) single stage method purification yolk antibody, ELISA measures antibody titer, and the yolk antibody that after the 4th immunity, ELISA measures is tired as 1:12560.
In step (4), spray-dired inlet temperature is 120 ℃-140 ℃, 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 there is classical symptom, heart, the liver lesion of the fugu obscurus in dead latter a hour carry out 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 on 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, has hemorrhage patch not of uniform size; Gall-bladder expands; Enteron aisle, without food, is full of spumescence mucus, and intestinal mucosa is hemorrhage.
The present invention is easy to operate, effective, and the yolk antibody that after the 4th immunity, ELISA measures is tired as 1:12560., the yolk antibody powder of further making is tired constant.Compare unique distinction of the present invention is also with other technologies: 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 that the Aeromonas veronii of take is prepared as antigen was not met report at home and abroad equally.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 invention will be further described.
The pathogenic Aeromonas veronii in fugu obscurus source, deposit number is 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
A preparation method for the pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source, comprises the following steps:
(1) by deposit number, be 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, in 37 ℃, cultivates 20h; Use aseptic PBS(0.01M, pH7.2) wash lower lawn, measuring and adjust bacterial concentration is 2.5 * 10
10cFU/mL; After pure inspection is qualified, press 0.5%(V/V) 37 ℃ of deactivation 24h of formalin solution, antigen is prepared in the qualified rear confession of steriling test; Every 5 weight part inactivated bacterial liquid add aluminium hydroxide gel 1 weight part of sterilizing, add that to be equivalent to the Thiomersalate of inactivated bacterial liquid and aluminium hydroxide gel gross weight 0.01% anticorrosion, obtain vaccine;
(2) immune programme for children: 28 week age, Luo Man laying hen carried out wing venous blood collection serum in contrast, opened and collected egg postpartum as experiment contrast; Open the 3rd day postpartum, the vaccine chest muscle multi-point injection immunity of preparing by step (1), dosage 0.5mL/ is only; Within after first immunisation 1 week, carry out immunity for the second time, immunizing dose 1.0mL/ only; For the second time after immunity every 20d booster immunization, continuous immunity 2 times, immunizing dose is 1.0mL/ only; 40d after the 4th immunity, then carry out immunity, immunizing dose is 2.0mL/; From first immunisation, latter 7 days wing venous blood collection separation of serum of each immunity, and collect all eggs, the egg of collecting at interval of 7d after immunity is carried out to 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 clear water grooming, isolate yolk under aseptic condition, add the physiological saline of 1.5 times of volumes, high-speed homogenization is prepared emulsion;
(4) preparation of powdery yolk: will spray through the pretreated yolk liquid of step (3) dry, and make powdery yolk, wherein the i.e. pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source of IgY.
Step (2) is to the egg of collecting at interval of 7d after immunity, under aseptic condition, isolate yolk, 80g/L PEG-8 000(PEG-8000) single stage method purification yolk antibody, ELISA measures antibody titer, and the yolk antibody that after the 4th immunity, ELISA measures is tired as 1:12560.In step (4), spray-dired inlet temperature is 120 ℃-140 ℃, 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 there is classical symptom, heart, the liver lesion of the fugu obscurus in dead latter a hour carry out 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 on 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, has hemorrhage patch not of uniform size; Gall-bladder expands; Enteron aisle, without food, is full of spumescence mucus, and intestinal mucosa is hemorrhage.
Claims (1)
1. a method of preparing pathogenic Aeromonas veronii yolk antibody, is characterized in that: comprise the following steps:
(1) by deposit number, be 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, in 37 ℃, cultivates 20h; With aseptic PBS, wash lower lawn, measuring and adjust bacterial concentration is 2.5 * 10
10cFU/mL; After pure inspection is qualified, by 37 ℃ of deactivation 24h of 0.5% formalin solution, antigen is prepared in the qualified rear confession of steriling test; Every 5 weight part inactivated bacterial liquid add aluminium hydroxide gel 1 weight part of sterilizing, add that to be equivalent to the Thiomersalate of inactivated bacterial liquid and aluminium hydroxide gel gross weight 0.01% anticorrosion, obtain vaccine;
(2) immune programme for children: 28 week age, Luo Man laying hen carried out wing venous blood collection serum in contrast, opened and collected egg postpartum as experiment contrast; Open the 3rd day postpartum, the vaccine chest muscle multi-point injection immunity of preparing by step (1), dosage 0.5mL/ is only; Within after first immunisation 1 week, carry out immunity for the second time, immunizing dose 1.0mL/ only; For the second time after immunity every 20d booster immunization, continuous immunity 2 times, immunizing dose is 1.0mL/ only; 40d after the 4th immunity, then carry out immunity, immunizing dose is 2.0mL/; From first immunisation, latter 7 days wing venous blood collection separation of serum of each immunity, and collect all eggs, the egg of collecting at interval of 7d after immunity is carried out to 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 clear water grooming, isolate yolk under aseptic condition, add the physiological saline of 1.5 times of volumes, high-speed homogenization is prepared emulsion;
(4) preparation of powdery yolk: will spray through the pretreated yolk liquid of step (3) dry, and make powdery yolk, wherein the i.e. pathogenic Aeromonas veronii yolk antibody in anti-fugu obscurus source of IgY;
Step (2), to the egg of collecting at interval of 7d after immunity, is isolated yolk under aseptic condition, 80g/L PEG-8 000 single stage method purification yolk antibody, and ELISA measures antibody titer, and the yolk antibody that after the 4th immunity, ELISA measures is tired as 1:12560;
In step (4), spray-dired inlet temperature is 120 ℃-140 ℃, and air outlet temperature is 68 ℃-72 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310233318.XA CN103304664B (en) | 2012-06-04 | 2012-06-04 | Method for preparing pathogenic aeromonas veronii egg yolk antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310233318.XA CN103304664B (en) | 2012-06-04 | 2012-06-04 | Method for preparing pathogenic aeromonas veronii egg yolk antibody |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210181107 Division CN102718866B (en) | 2012-06-04 | 2012-06-04 | Method for preparing takifugu obscurus source resistant pathogenic aeromonas veronii egg-yolk antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103304664A CN103304664A (en) | 2013-09-18 |
| CN103304664B true CN103304664B (en) | 2014-10-29 |
Family
ID=49130418
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310233318.XA Active CN103304664B (en) | 2012-06-04 | 2012-06-04 | Method for preparing pathogenic aeromonas veronii egg yolk antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103304664B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101074260A (en) * | 2007-03-20 | 2007-11-21 | 福建省农业科学院生物技术研究所 | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal |
| CN101993834A (en) * | 2010-05-20 | 2011-03-30 | 上海海洋大学 | Method for separating Aeromonas molluscorum producing tetrodotoxin from Takifugu fasciatus tissue and fermentation culture method of Aeromonas molluscorum as well as detection method of produced tetrodotoxin |
| CN102276720A (en) * | 2011-07-29 | 2011-12-14 | 江苏畜牧兽医职业技术学院 | High-biological-activity egg yolk antibody and preparation method thereof |
-
2012
- 2012-06-04 CN CN201310233318.XA patent/CN103304664B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101074260A (en) * | 2007-03-20 | 2007-11-21 | 福建省农业科学院生物技术研究所 | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal |
| CN101993834A (en) * | 2010-05-20 | 2011-03-30 | 上海海洋大学 | Method for separating Aeromonas molluscorum producing tetrodotoxin from Takifugu fasciatus tissue and fermentation culture method of Aeromonas molluscorum as well as detection method of produced tetrodotoxin |
| CN102276720A (en) * | 2011-07-29 | 2011-12-14 | 江苏畜牧兽医职业技术学院 | High-biological-activity egg yolk antibody and preparation method thereof |
Non-Patent Citations (6)
| Title |
|---|
| 吴同垒 等.维氏气单胞菌研究进展.《中国兽药杂志》.2011,第45卷(第7期), |
| 嗜水气单胞菌亚单位成分卵黄抗体在鳗鲡养殖中应用;庄政;《中国优秀硕士学位论文全文数据库》;20090615;D052-4 * |
| 庄政.嗜水气单胞菌亚单位成分卵黄抗体在鳗鲡养殖中应用.《中国优秀硕士学位论文全文数据库》.2009, |
| 抗嗜水气单胞菌卵黄抗体的制备及保护效果;曹军 等;《湖北农业科学》;20090630;第48卷(第6期);1443-1446 * |
| 曹军 等.抗嗜水气单胞菌卵黄抗体的制备及保护效果.《湖北农业科学》.2009,第48卷(第6期), |
| 维氏气单胞菌研究进展;吴同垒 等;《中国兽药杂志》;20111231;第45卷(第7期);41-44 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103304664A (en) | 2013-09-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105198988B (en) | Anti- Vibrio splindidus Yolk antibody and preparation method thereof | |
| CN104208673B (en) | A kind of fowl antiviral composition, lyophilized powder, preparation method and application | |
| CN101343320A (en) | Anti-vibrio parahaemolyticus chicken yolk antibody, preparation method and application thereof | |
| BR112021000100A2 (en) | ANTIBODIES AGAINST AGENTS CAUSING POULTRY DISEASE AND USES OF THE SAME | |
| CN101081866A (en) | Domestic animal and aquatic product causal agent resistant specific IgY or compound IgY and application thereof | |
| Xu et al. | Immunomodulatory effects of chicken egg yolk antibodies (IgY) against experimental Shewanella marisflavi AP629 infections in sea cucumbers (Apostichopus japonicus) | |
| CN102988971B (en) | Preparation method of porcine epidemic diarrhea virus genetic engineering subunit oral vaccine | |
| CN104001171A (en) | Preparation method of compound IgY for preventing various diarrheas of piglets | |
| CN105198989B (en) | Anti-yellowing sea Shewanella Yolk antibody and preparation method thereof | |
| CN101259272A (en) | Yolk antibody feed additive and injection for resisting porcine reproductive and respiratory syndrome and preparation thereof | |
| CN103495167B (en) | A kind of preparation method of chicken infection bursal disease composite live vaccine | |
| CN101690811B (en) | Concentrated freeze-dried yolk antibody composite preparation for infectious bursal disease and preparation process thereof | |
| Zhang et al. | Passive protection of Japanese pufferfish (Takifugu rubripes) against Vibrio harveyi infection using chicken egg yolk immunoglobulins (IgY) | |
| CN104072609B (en) | A kind of preparation method of Yolk antibody | |
| CN101259275A (en) | Yolk antibody feed additive and injection for resisting sheep virosis and preparation thereof | |
| CN102718866B (en) | Method for preparing takifugu obscurus source resistant pathogenic aeromonas veronii egg-yolk antibody | |
| CN102304182B (en) | Preparation method and use of enterotoxigenic Escherichia coli egg yolk antibody powder | |
| CN103304664B (en) | Method for preparing pathogenic aeromonas veronii egg yolk antibody | |
| CN101074260A (en) | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal | |
| CN100569285C (en) | The application of immunostimulating complex in preparation fish immunity preparation | |
| CN102908620A (en) | Preparation method of egg yolk antibody injection for treating piglet diarrhea | |
| CN101856495B (en) | Medical composition and mixture for treating chicken infectious bursal disease and preparation method thereof | |
| CN104031152A (en) | Recombined pig/cow source escherichia coli heat stable enterotoxin fusion protein STp5-His, monoclonal antibody for resisting protein and application of protein | |
| CN107118275B (en) | Fish IL-1 β -resistant egg yolk antibody and preparation method thereof | |
| CN102770452B (en) | An IgY for a PA-MSHA bacterial strain, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Cao Jun Inventor after: Wang Yongjun Inventor after: Chang Yan Inventor after: Jin Dayong Inventor after: Wang Zhaohui Inventor after: Liu Siyuan Inventor after: Xu Lei Inventor before: Cao Jun Inventor before: Chang Yan Inventor before: Jin Dayong Inventor before: Wang Zhaohui Inventor before: Liu Siyuan Inventor before: Xu Lei |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: CAO JUN CHANG YAN JIN DAYONG WANG ZHAOHUI LIU SIYUAN XU LEI TO: CAO JUN WANG YONGJUN CHANG YAN JIN DAYONG WANG ZHAOHUI LIU SIYUAN XU LEI |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191127 Address after: No.1, floor 3, No.319, zhanggongshan Road, Yuhui District, Bengbu City, Anhui Province Patentee after: Bengbu guijiu Intellectual Property Service Co., Ltd Address before: 226019 Jiangsu city of Nantong province sik Road No. 9 Patentee before: Nantong University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201019 Address after: 226000 No. 266 Century Avenue, hi tech Zone, Jiangsu, Nantong Patentee after: Nantong Yaoxiang Technology Co., Ltd Address before: No.1, floor 3, No.319, zhanggongshan Road, Yuhui District, Bengbu City, Anhui Province Patentee before: Bengbu guijiu Intellectual Property Service Co.,Ltd. |