CN102276720A - High-biological-activity egg yolk antibody and preparation method thereof - Google Patents
High-biological-activity egg yolk antibody and preparation method thereof Download PDFInfo
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- CN102276720A CN102276720A CN2011102156843A CN201110215684A CN102276720A CN 102276720 A CN102276720 A CN 102276720A CN 2011102156843 A CN2011102156843 A CN 2011102156843A CN 201110215684 A CN201110215684 A CN 201110215684A CN 102276720 A CN102276720 A CN 102276720A
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Abstract
The invention discloses a high-biological-activity egg yolk antibody and a preparation method thereof. In the antibody disclosed by the invention, aerolysin and extracellular proteases, which are main pathogenic factors of pathogenic aeromonas hydrophila, are respectively taken as immunogens to immunize laying hens simultaneously by a proper immunizing program, so that high-immunity eggs can be obtained with titer of 1:212; and the antibody is purified by an octanoic acid-ammonium sulfate method, and subjected to spray drying. According to the preparation method, the antibody has high titer, can be used for controlling bacterial diseases in fish, and has 90 percent of immunity protection effect to pathogenic fish with hemorrhagic disease. By adopting the method, the antibody can be produced in large scale; and the method has low cost, saves manpower and plant space, and has high social benefit and wide prospect.
Description
Technical field
The technology of preparing of a kind of anti-gas lysin and the composite intensified yolk antibody dry powder of extracellular protease is used for the fish bacterial disease control.Belong to biological products and industrial separation technical field.
Background technology
Aeromonas hydrophila (Aeromonas hydrophila) is the bacterium of being everlasting that a class extensively is present in water surrounding, it is the main pathogen of bacteriosises such as China's cultured freshwater fish fulminant hemorrhagic disease, wide in the aquatic products production process by many, the popular scopes of its disease that causes harm kind, mortality ratio is high, breed brings greater loss to aquatic products.Traditional remedies is to use antimicrobial drug, cooperates Chinese medicine preparation sometimes.But the use of antimicrobial drug not only brings serious resistance problem, and easily produces residual in fishery products.Therefore, seek a kind of safe, the antimicrobial drug substitute becomes a problem demanding prompt solution efficiently.Special yolk antibody (Egg yolk immunoglobulin, IgY) can treat the disease that causes by bacterium or virus infection, have safe noresidue, do not produce resistance, advantage such as cheap and easy to get, be a kind of microbiotic substitute with broad prospect of application.
At present, yolk antibody development technology is just becoming the new focus of domestic and international field of medicaments.At home, about the application existing a lot of reports of yolk antibody in the multiple infectious diseases control of animal, but only find 2 pieces in the correlative study of fish disease aspect preventing and treating about special yolk antibody, (2006) such as Dalian aquatic product Academy Wang Bin are separated in the turbot body and are obtained Edwardsiella tarda and make deactivation vaccine, obtain special yolk antibody behind the immunity laying hen, the malicious turbot of attacking against each other has obvious prevention effect.(2006, Master's thesis) such as the Lei Yong of Agricultural University Of Hunan have been developed antiviral yolk antibody and have been used for treating the shrimp leukodermia, and injecting pathway is invalid, but the prevention of oral antibody prawn white spot disease and treatment all have obvious provide protection.Abroad yolk antibody also there is research in the application of fish bacterial disease aspect preventing and treating.Hatta etc. have reported the special yolk antibody that is used to prevent and treat eel; The yolk antibody powder of the anti-tarda of the eight Tian Yiyong eel of throwing something and feeding, the result proves the tarda disease that can prevent eel fully; Arastech etc. carry out the passive immunization prophylactic tria with yolk antibody to rainbow trout, after the discovery rainbow trout is taken yolk antibody 15d, Vibrio anguillarum have been produced tangible immunizing power; Gutierrez etc. carry out oral immunity with yolk antibody to Japanese eel, find that the quantity of Edwardsiella 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.But the yolk antibody of the main pathogenic bacterium at fish fulminant hemorrhagic disease---Aeromonas hydrophila does not still have open production technology at present.
The main virulence factor Aer toxin of Aeromonas hydrophila is the main virulence factor of present internationally recognized Aeromonas hydrophila, and its gene structure has higher homology; Extracellular protease (ECPase) is one of Aeromonas hydrophila excretory extracellular products, is another kind of main virulence factor, has good antigen provide protection, and the ECPase gene order of different sources has high homology.
The applicant finds by test, the gas lysin Aer of Aeromonas hydrophila different strains and extracellular protease ECPase can intersect respectively and suppress haemolysis and molten albumen test, can remedy between the bacterial strain almost can not cross reaction defective, and further strengthen the effect of IgY, and can not suppress production of antibodies mutually between between the three, can both produce higher level antibody.
Summary of the invention
The object of the invention is to provide a kind of yelk antibody with high bioactivity, be used to prevent and treat fish bacterial disease especially fish fulminant hemorrhagic disease have unusual effect.
Another object of the present invention provides the preparation method of this yelk antibody with high bioactivity of preparation.
The objective of the invention is to realize in the following manner:
A kind of yelk antibody with high bioactivity, this antibody is distinguished immunization laying hen by Aeromonas hydrophila gas lysin and extracellular protease as immunogen, and collects by the egg that immune chicken produced, and separates obtaining yelk antibody with high bioactivity from this egg.
Describedly prepare by following steps: the Aeromonas hydrophila culture supernatant as immunogenic Aeromonas hydrophila gas lysin, ammoniumsulphate soln fractionation precipitation through 20%～60% saturation ratio, refrigeration is spent the night, centrifugal (preferred 5000rpm is centrifugal), with pH=7.8～8.0, dissolving of 50mmol/L Tris-HCl damping fluid and dialysis; Centrifugal (preferred 12000rpm is centrifugal), supernatant is adjusted protein concentration to 100～200 μ g/mL.Can be fully emulsified when immunity is used with the equivalent freund's adjuvant.
Describedly prepare by following steps: the Aeromonas hydrophila culture supernatant as immunogenic extracellular protease, ammoniumsulphate soln precipitation with 50%～100% saturation ratio, refrigeration is spent the night, with pH=7.8～8.0, and dissolving of 50mmol/L Tris-HCl damping fluid and dialysis; Centrifugal (preferred 12000rpm is centrifugal), supernatant is adjusted protein concentration to 100～200 μ g/mL.Can be fully emulsified when immunity is used with the equivalent freund's adjuvant.
Described laying hen immunity is with Aeromonas hydrophila gas lysin and two kinds of immunogens difference of extracellular protease multi-point injection laying hen, each minute three immunity, and each immunity was at least 2 weeks pitch time, the each 0.5～3mL/ of immunizing dose is only.
The method of separating yolk antibody adopts sad-ammonium sulfate method to separate and obtains yolk antibody.Described employing is sad-and the ammonium sulfate method separates and obtains the yolk antibody concrete steps and be: and the yolk after the immunity mixes with the pH5.0 sodium-acetate buffer earlier, adds 4% n-caprylic acid, and mixed at room temperature refrigerates and leaves standstill more than the 2h, and it is fully precipitated; Sad post precipitation suction filtration, suction filtration liquid is centrifugal, collects supernatant, adds equal-volume saturated ammonium sulphate solution under the condition of ice bath, makes the antibody precipitation, and is centrifugal, collects yolk antibody.
Can carry out drying to yolk antibody after separating yolk antibody, preferably adopt spray-drying process, inlet temperature is 130～140 ℃, and temperature out is 70～90 ℃.
The preparation method of above-mentioned yelk antibody with high bioactivity distinguishes immunization laying hen with Aeromonas hydrophila gas lysin and extracellular protease as immunogen, and collects by the egg that chicken produced of immunity, separates obtaining yelk antibody with high bioactivity from this egg.
Yolk antibody of the present invention can be used in the medicine of preparation control fish bacterial disease, is particularly useful for preparing the medicine of control fish fulminant hemorrhagic disease.During use, can digestive tube administration or parenteral administration will be passed through after the antibody spice mixed feeding.
The used Aeromonas hydrophila suitable culture base of the present invention is nutrient agar medium or nutrient broth, and culture temperature is preferably 28～30 ℃.Described Aeromonas hydrophila gas lysin fractionation precipitation extracting method is preferred earlier with the ammoniumsulphate soln precipitation foreign protein of 20% saturation ratio, and the ammoniumsulphate soln with 60% saturation ratio precipitates gas lysin Aer again; At last with dissolving of 50mmol/L Tris-HCl damping fluid and dialysis.After the extracting method of extracellular protease ECPase preferably adopts the ammoniumsulphate soln deposit E CPase of 70% saturation ratio, again with 50mmol/L Tris-HCl dissolving and dialysis.The extraction of yolk antibody, purifying preferred version are to precipitate foreign protein with 4% n-caprylic acid earlier, use the saturated ammonium sulphate purified solution of 50% saturation ratio again.
Immunogen of the present invention is gas lysin Aer and extracellular protease, immune programme for children is two kinds of immunogens immune laying hens respectively, and route of inoculation is chest muscle or subcutaneous multi-point injection, fundamental immunity once after booster immunization secondary again, be at least 2 weeks pitch time, preferred each 2 weeks at interval.Immunizing dose only is advisable with each 0.5～3mL/, and three times immunizing dose can increase progressively successively.Egg, antibody purification can be collected in one week in last immunity back.The commercially available Aeromonas hydrophila that the present invention adopts all can be used.
Beneficial effect of the present invention compared with the prior art: with compare with the Aeromonas hydrophila vaccine immunization; adopt Aeromonas hydrophila main virulence factor gas lysin Aer and extracellular protease ECPase respectively as immunogen preparing IgY; not only overcome the influence of Aeromonas hydrophila serotype antagonist protection effect better, and the antibody titer that obtains is higher.The plant and instrument that uses in the antibody powder production process is simple, and agents useful for same is cheap and easy to get, and is pollution-free, the antibody titer height of acquisition, and immunocompetence is strong, and is good to the prevention effect of fish fulminant hemorrhagic disease, do not produce drug residue, can ensure the safety of fishery products.In addition, the production model of high-immunity egg can adopt the form of signing an agreement with the breeding layer chicken family, utilize the technology of the present invention with laying hen as bio-reactor, the input and the risk of breeding layer chicken link had both been avoided, obtain a large amount of high-immunity eggs in low-cost mode, easy to implement, the social benefit height.
Description of drawings
The SDS-PAGE collection of illustrative plates of Fig. 1 embodiment 2 gas lysin Aer (gas lysin Aer is about 45kDa)
The SDS-PAGE collection of illustrative plates (ECPase is about 35kDa) of Fig. 2 embodiment 3 extracellular protease ECPase
4 two kinds of immunogens of Fig. 3 embodiment are the immune yolk antibodies change curve in time of tiring simultaneously
The SDS-PAGE collection of illustrative plates of yolk antibody (3 and 4 is 2 kinds of yolk antibodies that sad-ammonium sulfate method is purified) behind the purifying among Fig. 4 embodiment 5
Embodiment
The cultivation of embodiment 1 Aeromonas hydrophila F8 (provide by Shanghai Ocean University, separate) from grass carp
With agar plate or nutrient broth (pH7.0～7.5) inoculation Aeromonas hydrophila, in 28～30 ℃ of cultivations, inoculation back 6～18h is a logarithmic phase.Colonial morphology is smooth, neat in edge, and surface wettability, circle, dimpling have special aromatic odour, and white is to faint yellow.
The extraction of embodiment 2 Aeromonas hydrophila gas lysins and the preparation of oil seepage
Aeromonas hydrophila is cultivated based on 28～30 ℃ with LB and cultivates 48～60h, 4500rpm centrifuging and taking supernatant, ammonium sulfate with 20% saturation ratio precipitates 4h at 4 ℃ earlier, 5000rpm is centrifugal, get supernatant, use the ammonium sulfate precipitation of 60% saturation ratio again, 4 ℃ are spent the night, 5000rpm is centrifugal, dissolves and dialyses the centrifugal 5min of 12000rpm two days with 50mmol/L Tris-HCl (pH=7.8～8.0), decon, supernatant is adjusted protein concentration to 100 μ g/mL, and is fully emulsified with the freund's adjuvant of supernatant equivalent, do not disperse to get final product in the 1min of back to splashing in the water.
The extraction of embodiment 3 extracellular protease ECPase and the preparation of oil seepage
Aeromonas hydrophila is cultivated based on 28～30 ℃ with LB and cultivates 48～60h, 4500rpm is centrifugal, get supernatant, with the ammonium sulfate precipitation of 70% saturation ratio, 4 ℃ are spent the night, 5000rpm is centrifugal, dissolve and dialysed the centrifugal 5min of 12000rpm, decon two days with 50mM Tris-HCl (pH7.8～8.0), supernatant is adjusted protein concentration to 200 μ g/mL, and is fully emulsified with the freund's adjuvant of supernatant equivalent.
25 the week age Luo Man laying hen, breeding observing is after 2 weeks, the gas lysin Aer that will obtain by the described method of the foregoing description 1-3 and two kinds of oil seepages of extracellular protease ECPase are chest muscles or subcutaneous multi-point injection respectively, each minute three immunization laying hens, each immunity pitch time was 2 weeks.Immunizing dose be followed successively by 0.5mL/ only, 1mL/ only, 2mL/ only.Last immunity back began to collect egg in one week, adopted indirect elisa method to measure yolk antibody and tired.Head exempts from back different time yolk antibody and tires as table 1 and Fig. 3.
It is the square formation volumetry that indirect ELISA is measured the yolk antibody method of tiring.Base program is: enzyme plate 1～8 row successively with 10,5,2.5,1.25,0.625,0.31,0.15mg/mL gas lysin Aer or extracellular protease ECPase and coating buffer bag quilt, 4 ℃ are spent the night; Washing pats dry, and repeats 3 times; Every hole adds i%OVA 200 μ L, 37 ℃ of sealing 60min; Wash 4 times, pat dry; Every hole adds the yolk antibody liquid to be measured (yolk liquid is by implementing gained among the embodiment 5) of the different extension rates of 100 μ L, 37 ℃ of incubation 60min; Repeated washing 4 times pats dry; Add goat-anti chicken HRP-IgG (1: 5000), 100 μ L/ holes, 37 ℃ of incubation 60min; Wash 5 times; Every hole adds TMB-H
2O
2100 μ L, 37 ℃ of lucifuge reaction 15min; Every hole adds 50 μ LH
2SO
4(2mol/L), termination reaction is surveyed the OD value at the 450nm place.With the OD value near 1 and jump the maximum pairing antibody dilution in hole as antibody titer with peripheral hole.
Table 1 head exempts from back different time yolk antibody and tires
Extraction, the purifying of embodiment 5 yolk antibodies
Mix with 2 times of volume pH5.0 sodium-acetate buffers earlier according to the yolk of obtaining after the embodiment 4 laying hen immunity.Dropwise add 4% n-caprylic acid, mixed at room temperature 30min under the stirring at room.4 ℃ leave standstill more than the 2h, and it is fully precipitated.Three layers of gauze suction filtration, the centrifugal 10min of suction filtration liquid collects supernatant.The saturated ammonium sulphate that adds equal-volume 50% saturation ratio under the condition of ice bath makes the antibody precipitation.Centrifugal, collect antibody.With PBS (0.01mol/L, pH7.4) dialysis 24h, during change dialyzate 3～5 times.With conventional sample diluting liquid yolk liquid is diluted to volume before the purifying.Identify (as accompanying drawing 4) by the SDS-PAGE electrophoresis, indirect ELISA is measured it and is tired.
Antibody liquid dry technology behind embodiment 6 yolk antibody liquid or the purifying
Yolk liquid behind extraction, the purifying enters kiln through nozzle atomization, with being yolk powder after the hot air dryer drying.Inlet temperature generally is set at 130～140 ℃, and the temperature of outlet is 70～90 ℃.
The immanoprotection action of embodiment 7 yolk antibodies
50 of crucians are divided into 5 groups, 10/group, are made as 4 different tests groups and 1 blank group respectively.The test group preceding 1～2h that feeds intake every day is sprayed onto yolk liquid (measuring protein concn behind sad-ammonium sulfate method purifying) on the feed, makes that protein and feed weight ratio are respectively 0.4mg/g, 0.2mg/g, 0.1mg/g, 0.05mg/g in the yolk liquid.The control group feed sprays the egg yolk liquid 0.2mg/10g of blank immune group laying hen.Feed intake by fish body weight 2% every day, and all backs are with minimum lethal dose viable bacteria (10
8The CFU/ bar) bacterium is attacked in intramuscular injection, changes not containing the feedstuff feeding of yolk antibody, observes and the death condition of fish in the week attacked behind the bacterium in record, and dissects and the separating again of cause of disease, attacks to determine whether to die from that malicious viable bacteria causes.Experiment finishes the back and calculates immune protective rate.Immune protective effect such as table 2.
The attack against each other immunoprophylaxis effect of bacterium crucian of table 2 yolk antibody
The yolk antibody powder is by the spice mixed feeding; dosage is in antibody and quality of the fodder ratio; with 0.05mg/g dosage mixed feeding one week of administration; the death time that Aeromonas hydrophila infects the disease fish can obviously be prolonged; with 0.1mg/g dosage mixed feeding one week of administration; the immune protective rate that Aeromonas hydrophila is infected can reach 30%; with 0.2mg/g, 0.4mg/g dosage mixed feeding one week of administration; the immune protective rate that Aeromonas hydrophila is infected can reach 70% and 90% respectively, and Aeromonas hydrophila is infected prophylactic effect preferably.
Therapeutic test for yolk antibody, throw something and feed with the antibody spice again after bacterium causes the fish morbidity because of attacking earlier, substantially do not ingest after found that the morbidity of test fish, cause antibody can't enter in the disease fish body, therefore the therapeutic test of yolk antibody ends in failure, and the treatment of pointing out this yolk antibody to be used for the disease fish in production practice can be used parenteral administration instead.
Claims (10)
1. yelk antibody with high bioactivity, it is characterized in that this antibody by Aeromonas hydrophila gas lysin and extracellular protease as immunogen immunization laying hen respectively, and collect by the egg that immune chicken produced, from this egg, separate obtaining yelk antibody with high bioactivity.
2. yolk antibody according to claim 1, it is characterized in that describedly preparing by following steps: the Aeromonas hydrophila culture supernatant as immunogenic Aeromonas hydrophila gas lysin, ammoniumsulphate soln fractionation precipitation through 20%～60% saturation ratio, refrigeration is spent the night, centrifugal, with pH=7.8～8.0, dissolving of 50mmol/L Tris-HCl damping fluid and dialysis; Centrifugal, supernatant is adjusted protein concentration to 100～200 μ g/mL.
3. yolk antibody according to claim 1, it is characterized in that describedly preparing by following steps: the Aeromonas hydrophila culture supernatant as immunogenic extracellular protease, ammoniumsulphate soln precipitation with 50%～100% saturation ratio, refrigeration is spent the night, with pH=7.8～8.0, dissolving of 50mmol/L Tris-HCl damping fluid and dialysis; Centrifugal, supernatant is adjusted protein concentration to 100～200 μ g/mL.
4. yolk antibody according to claim 1, it is characterized in that described laying hen immunity is with Aeromonas hydrophila gas lysin and two kinds of immunogens difference of extracellular protease multi-point injection laying hen, each immunity three times, each immunity was at least 2 weeks pitch time, the each 0.5～3mL/ of immunizing dose is only.
5. yolk antibody according to claim 1 is characterized in that the method for separating yolk antibody adopts sad-ammonium sulfate method separation to obtain yolk antibody.
6. yolk antibody according to claim 5, it is characterized in that described employing sad-the ammonium sulfate method separates and to obtain the yolk antibody concrete steps and be: the yolk after the immunity mixes with the pH5.0 sodium-acetate buffer earlier, add 4% n-caprylic acid, mixed at room temperature, refrigeration is left standstill more than the 2h, and it is fully precipitated; Sad post precipitation suction filtration, suction filtration liquid is centrifugal, collects supernatant, adds equal-volume saturated ammonium sulphate solution under the condition of ice bath, makes the antibody precipitation, and is centrifugal, collects yolk antibody.
7. yolk antibody according to claim 1 carries out drying to yolk antibody after it is characterized in that separating yolk antibody, adopts spray-drying process, and inlet temperature is 130～140 ℃, and temperature out is 70～90 ℃.
8. the preparation method of a yelk antibody with high bioactivity, it is characterized in that this method is that Aeromonas hydrophila gas lysin and extracellular protease are distinguished immunization laying hen as immunogen, and collect by the egg that chicken produced of immunity, from this egg, separate obtaining yelk antibody with high bioactivity.
9. the application of the described yolk antibody of claim 1 in the medicine of preparation control fish bacterial disease.
10. the application of the described yolk antibody of claim 1 in the medicine of preparation control fish fulminant hemorrhagic disease.
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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 |
| TWI460189B (en) * | 2012-08-01 | 2014-11-11 | Univ Nat Taiwan Normal | AN EXTRACTION METHOD FOR γ-LIVETIN (IGY) FROM YOLK |
| CN107164348A (en) * | 2017-04-11 | 2017-09-15 | 大连民族大学 | Aeromonas hydrophila surface specific antigen A hsA and its antibody and application |
-
2011
- 2011-07-29 CN CN2011102156843A patent/CN102276720A/en active Pending
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| 吴媛媛等: "抗氯霉素卵黄抗体的制备及其分离纯化", 《过程工程学报》 * |
| 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农业科技辑》 * |
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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 |
| TWI460189B (en) * | 2012-08-01 | 2014-11-11 | Univ Nat Taiwan Normal | AN EXTRACTION METHOD FOR γ-LIVETIN (IGY) FROM YOLK |
| US9605052B2 (en) | 2012-08-01 | 2017-03-28 | National Taiwan Normal University | Method for extracting IgY (γ-livetin) from egg yolk |
| CN107164348A (en) * | 2017-04-11 | 2017-09-15 | 大连民族大学 | Aeromonas hydrophila surface specific antigen A hsA and its antibody and application |
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Application publication date: 20111214 |