CN100404553C - Sturgeon family fish ovovitellin preparation method and uses - Google Patents
Sturgeon family fish ovovitellin preparation method and uses Download PDFInfo
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- CN100404553C CN100404553C CNB2005100104213A CN200510010421A CN100404553C CN 100404553 C CN100404553 C CN 100404553C CN B2005100104213 A CNB2005100104213 A CN B2005100104213A CN 200510010421 A CN200510010421 A CN 200510010421A CN 100404553 C CN100404553 C CN 100404553C
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Abstract
The present invention relates to a preparation method and the application of ovovitellin of acipenseridae fish stocks, which relates to a preparation method of the ovovitellin for identifying the male and female of acipenseridae fish stocks and a method for identifying the male and female of acipenseridae fish stocks by using the ovovitellin. No immunological method for detecting the vitellogenin of acipenseridae fish stocks by using the ovovitellin of acipenseridae fish stocks is established in China, and only a few enzyme-linked immunosorbent methods for acipenseridae fish stocks are established in foreign countries. The preparation method of the present invention comprises the following steps: firstly, ova of acipenseridae fish stocks are used; after the ova are homogenized at a high speed at a low temperature, and are centrifugated at a low temperature, the ova are precipitated by saturated ammonium sulfate and are separated and purified by gel columns; after the ova are dialyzed and desalted, ovovitellin freeze-dried powder is prepared; the freeze-dried powder is weighed, and is dissolved in physiological saline; Freund's adjuvant is added to the physiological saline for emulsifying the freeze-dried powder; rabbits are immunized for preparing polyclonal antibodies. The present invention can be used for identifying the male and female of acipenseridae fish stocks, and has the advantages of strong specificity, high sensitivity, convenience, rapidness, low cost, etc.
Description
Technical field
The present invention relates to the preparation method of sturgeon family fish ovovitellin antibody and pass through to use vitellin antibody to differentiate the method for sturgeon section fish male and female.
Background technology
Sturgeon " roe sauce " is the very high food of nutritive value, is known as " black gold ", and market value is up to 2000～4000 dollars/kilogram.But the sturgeon life cycle is long, and sexual maturity evening (more than 9 years) is even the sex of ripe sturgeon also is difficult to from identification in appearance.For produce sturgeon seed sauce, people have to sturgeon cultivation after sexual maturity, just can identify male and female.In the cultivation time that reaches more than 9 years, because the closely milter of half is wherein arranged, production cost need double, and has wasted a large amount of funds and throughput (area).Therefore, explore sturgeon sex discrimination method, become " roe sauce " producer's consistent cry.
Vitellogenin is a kind of protein that is present in specifically in the oviparity jenny blood, go into ovum by the vitellogenin of hepatic secretion after, under the effect of enzyme, be degraded to vitellin(Vt).Vitellin is the major protein composition of ovocyte and important nutrition and the energy derive that fish embryo is grown as the degraded product of fish vitellogenin, and its accumulation is crucial to the growth of ovocyte.Vitellogenin and vitellin structurally have similarity, can detect by immunologic method.And do not have vitellogenin in the milter blood of under home, living, have or not vitellogenin in the sturgeon section fish blood as long as determine in the present invention, just its sex as can be known.
At present, the domestic detection method of also not using the fish vitellin to set up fish and sturgeon section fish vitellogenin, the external enzyme connection detection method of also only setting up minority sturgeon section fish is (as " people such as .Naoshi Hiramatsu is to Vitellogenin (Vg) and the vitellin(Vt) YP1 of hybrid sturgeon (Huso huso x Acipencer ruthenus), and YP2 and YP3 carries out the basic researchs in aspect such as molecular weight.Vitellogenin-derived yolk proteinsin a hybrid sturgeon, bester (Huso huso x Acipencer ruthenus): Identification, characterization and course of proteolysis during embryogenesis.ComparativeBiochemistry and Physiology-Part A:Molecular﹠amp; Integrative Physiology.2002,131 (2), 429-441. " and " people such as Grant W.Feist has set up the Vg euzymelinked immunosorbent assay (ELISA) influences .Evidence of Detrimental Effects ofEnvironmental Contaminants on Growth and Reproductive Physiology of WhiteSturgeon in Impounded Areas of the Columbia River.Environ HealthPerspect, 113 (12) to paddlefish Acipenser transmontanus to environmental pollutants; Dec 2005. "), and all be the detection of application foundation experiment and environmental classes hormone hormone.
Summary of the invention
In view of to the domestic detection method of also not using the fish vitellin to set up fish and sturgeon section fish vitellogenin, the external enzyme connection detection method of only setting up minority sturgeon section fish, and be not that the male and female that are applied to sturgeon section fish are differentiated, the present invention aims to provide a kind of preparation method of sturgeon family fish ovovitellin antibody and uses this technology and carry out the fish sex discriminating of sturgeon section.For achieving the above object, the present invention prepares sturgeon family fish ovovitellin antibody by the following technical solutions:
One, the preparation of sturgeon flying fish vitellin extracting solution:
1. take by weighing the Tris-HCl damping fluid of 0.02mol/l, the pH=8.0 of the fish-egg 5g of sturgeon section adding 15mL precooling in advance, high-speed homogenization, precipitation is abandoned in centrifugation, stays supernatant liquor;
2. supernatant liquor is by gel chromatographic columns wash-out sphadex G-150;
3. collect the elutriant that contains vitellin, the dialysis desalination obtains the vitellin extracting solution;
Two, sturgeon flying fish vitellin Polyclonal Antibody Preparation:
1. be lyophilized powder with the lyophilize of fish-egg yellow phosphorus protein extract, the vitellin lyophilized powder that takes by weighing 1mg is dissolved in the 1ml physiological saline, adds the 1ml Freund's complete adjuvant, emulsification under the low temperature, and multi-point injection under the animal skin carries out initial immunity;
2. the 28th, 38, the 48 day subcutaneous multiple spot booster immunization in each back once, immunizing dose adopts Freund's incomplete adjuvant emulsification antigenic solution with for the first time;
3. after 55 days, animal ear edge extracts small amounts of blood and measures antibody titer;
4. the heart blood sampling prepares sturgeon flying fish vitellin antiserum(antisera).
The present invention directly extracts vitellin from fish-egg, substitutes by the synthetic vitellogenin of estrogen-induced fish liver, and then the method for from blood, extracting.The sturgeon flying fish vitellin antibody of present method preparation can be used to detect the sex of sturgeon section fish; Based on immunology principle, can adopt double immunodiffusion, euzymelinked immunosorbent assay (ELISA) and immune colloid gold method to detect respectively, three kinds of detection side's ratio juris are:
(1) two-way immunodiffusion(ID) principle, promptly antigen, antibody can generate immunoprecipitation complex after spreading and meeting in certain medium, form macroscopic immunoprecipitation line in medium.
(2) enzyme linked immunological absorption detects principle, be the standard antigen non-specific adsorption after on the enzyme plate, seal unnecessary binding site, add serum to be checked and enzyme labelled antibody, after removing unnecessary sample to be checked and enzyme labelled antibody, add substrate and begin enzymatic reaction, and measure solution absorbency, absorbancy becomes negative correlation with vitellin(Vt) original content in the sample to be checked.Male fish have 100% color reaction, the colour developing of raun then a little less than.
(3) principle of immune colloid gold quick detection test paper bar is that specific antibody is fixed on the nitrocellulose membrane with ribbon, and colloid gold label reagent is adsorbed on the pad.Be added on the sample pad of test strip one end when testing sample serum after, move forward by wicking action, react to each other behind the colloid gold label reagent on the dissolving cured pad, when moving to sessile antibody regional again, the specificity combination takes place again with it and is trapped in the mixture of determinand and gold marked reagent, accumulate in to detect and be with, by the colloid gold label thing that can the estimate result that developed the color intuitively.
In method provided by the invention, easy to use, simple and easy, cheap double immunodiffusion is arranged; Have and detect accurately, operate complicated euzymelinked immunosorbent assay (ELISA); Immune colloid gold fast detection method easy to use in addition, that detection is quick, moderate.Aforesaid method not only can qualitative identification sturgeon section fish sex, also can the multiple sturgeon of quantitative detection section fish blood in and the content of vitellogenin in the tissue.
The present invention can detect having or not of vitellogenin in the sturgeon section fish serum sensitive, exactly, differentiates the sex of sturgeon section fish.Compare with the fish sex authentication method that hormone detection technique, anatomy method, ultrasound examination etc. are traditional, present method has high specificity, highly sensitive, convenient and swift, low cost and other advantages, and little to being examined the fish damage, be to carry out the Perfected process that the sturgeons male and female are differentiated.
Embodiment
Embodiment one: present embodiment prepares sturgeon family fish ovovitellin antibody according to following method:
One, the preparation of sturgeon flying fish vitellin extracting solution:
1. take by weighing the Tris-HCl damping fluid of 0.02mol/l, the pH=8.0 of the fish-egg 5g of sturgeon section adding 15ml precooling in advance, high-speed homogenization, precipitation is abandoned in centrifugation, stays supernatant liquor;
2. supernatant liquor is by the gel chromatographic columns wash-out;
3. collect the elutriant that contains vitellin, the dialysis desalination obtains the vitellin extracting solution;
Two, sturgeon flying fish vitellin Polyclonal Antibody Preparation:
1. be lyophilized powder with the lyophilize of fish-egg yellow phosphorus protein extract, the vitellin lyophilized powder that takes by weighing 1mg is dissolved in the 1ml physiological saline, adds the 1ml Freund's complete adjuvant, emulsification under the low temperature, and the subcutaneous multi-point injection of back part of animal carries out initial immunity;
2. the 28th, 38, the 48 day subcutaneous multiple spot booster immunization in each back once, immunizing dose adopts Freund's incomplete adjuvant emulsification antigenic solution with for the first time;
3. after 55 days, animal ear edge extracts small amounts of blood and measures antibody titer;
4. the heart blood sampling prepares sturgeon flying fish vitellin antiserum(antisera).
Embodiment two: present embodiment differentiates that with double immunodiffusion sturgeon section fish male and female are example, and it comprises the steps:
The separation and purification of A, vitellin standard substance:
1. the preparation of vitellin(Vt) extracting solution: take by weighing the Tris-HCl damping fluid 15ml that fish-egg 5g adds 0.02mol/l, the pH=8.0 of precooling in advance, the 10000rpm/min high-speed homogenization, 4 ℃, the centrifugal 20min of 12000rpm/min abandon precipitation, stay supernatant;
2. supernatant passes through gel chromatographic columns, filler is sephadex G-100, chromatography column adds Tris-HCl damping fluid (contain 2%NaCl and the 0.015% sodium azide) wash-out of 1mL supernatant liquor with 0.02mol/l, pH=8.0 with Tris-HCl damping fluid (containing 2%NaCl and the 0.015% sodium azide) balance of 0.02mol/l, pH=8.0;
3. collect the elutriant that contains vitellin, behind the dialysis desalination, lyophilize is a lyophilized powder.
B, vitellin Polyclonal Antibody Preparation:
1. take by weighing 1mg vitellin lyophilized powder, be dissolved in the 1ml physiological saline, add the Freund's complete adjuvant of 1ml, emulsification under the low temperature, the subcutaneous multi-point injection in White Rabbit back carries out initial immunity;
2. the 28th, 38, the 48 day subcutaneous multiple spot booster immunization in each back once, immunizing dose adopts Freund's incomplete adjuvant emulsification antigenic solution with for the first time;
3. after 55 days, rabbit ear edge extracts small amounts of blood and measures antibody titer;
4. the heart blood sampling prepares the vitellin antiserum(antisera).
C, double immunodiffusion detect:
1. get among Tris-HCl damping fluid (containing 2%NaCl and the 0.015% sodium azide) 100ml that agarose 1g is dissolved in 0.02mol/l, pH=8.0, aseptic pouring in the culture dish, after the cooling, the punch tool punching;
2. the empty antiserum(antisera) that adds in the middle of, the hole adds serum to be checked on every side, hatches check result 5 hours for 37 ℃;
3. the result judges: it is female that being of white precipitate line appears in serum to be checked hole and antiserum(antisera) hole, and what precipitation line do not occur is male.
Embodiment three: present embodiment differentiates that with euzymelinked immunosorbent assay (ELISA) sturgeon section fish male and female are example, and it comprises the steps:
The separation and purification of A, vitellin: same double immunodiffusion.
B, vitellin Polyclonal Antibody Preparation: same double immunodiffusion.
The preparation of C, enzyme labelled antibody:
1. taking by weighing HRP (horseradish peroxidase) 25mg, to be dissolved in volumetric concentration be in 1.25% the glutaraldehyde solution, in the room temperature standing over night;
2. reacted enzyme solution is used the physiological saline wash-out through the SephadexG-25 chromatography column, and flow rate control was collected brown effluent liquid at 1ml/l minute, greater than 5ml, then was concentrated into 5ml with PEG as volume; Place in the 25ml small beaker, slowly stir;
3. antibody 12.5mg to be marked is diluted to 5ml with physiological saline, dropwise adds in the enzyme solution under stirring;
4. use the carbonic acid buffer 0.25ml of 1mol/l, pH=9.5, continue to stir 3 hours;
5. add 0.2mol/l Methionin 0.25ml, behind the mixing, put room temperature 2 hours;
6. under agitation dropwise add the equal-volume saturated ammonium sulphate, put 4 ℃ 1 hour;
7. mixed solution is abandoned supernatant liquor in 3000rpm/min centrifugation half an hour, and throw out is dissolved in phosphoric acid-phosphate buffered saline buffer of 0.15mol/l, pH=7.4 with semi-saturation ammonium sulfate washed twice, last throw out;
8. above-mentioned solution is packed in the dialysis tubing, to phosphoric acid-phosphate buffered saline buffer dialysis of 0.15mol/l, pH=7.4, behind the removal ammonium ion, removed precipitation in centrifugal 30 minutes at 10000rpm/min, supernatant liquor is enzyme conjugates, after the packing, and stored frozen.
D, euzymelinked immunosorbent assay (ELISA) are differentiated sturgeon section fish male and female:
1. add vitellin standard substance coated elisa plate in the every hole of 96 hole enzyme plates, 4 ℃ of refrigerator bags are by the 24h that spends the night;
2. discard the liquid in the enzyme plate, every hole adds 360 μ l confining liquids, places 4 ℃ of refrigerators to seal the 24h that spends the night;
3. discard confining liquid, every hole adds 300 μ l washingss, slightly shake 1min after, discard washings, repeat 3 times;
4. every hole adds serum to be checked and enzyme labelled antibody, behind the incubated at room 1h, discards enzyme labelled antibody, washs 4 times;
5. every hole adds 100 μ l chromogenic substrates, and room temperature dark place reaction 20 minutes adds 50 μ l reaction terminating liquids, termination reaction;
6. according to having or not color reaction to judge the male and female of sturgeon section fish: on white background, the result that directly detects by an unaided eye, color is dark more in the reacting hole, and positive degree is strong more, is female sturgeon; Negative reaction is colourless or extremely shallow, is milter.
The designed direct competition method test kit of present embodiment is composed as follows:
Bag is by the 96 hole enzyme plates (1) of good standard antigen;
One of negative control (200 μ l/ prop up);
Enzyme mark vitellin antibody 50 μ l, dilution is 1000 times before using;
Respectively one of colour developing liquid, diluent and lavation buffer solution;
Stop buffer (0.5mol/L H
2SO
4) 8ml.
Annotate: diluent: the PBS damping fluid, PH 7.4; Washings: the diluent that contains 0.05%Tween20; Confining liquid: contain 1% bovine serum albumin diluent; Colour developing liquid: o-phenylenediamine solution (OPD).
Embodiment four: present embodiment differentiates that with immune colloidal gold fast detecting test paper strip method sturgeon section fish male and female are example, and it comprises the steps:
The separation and purification of A, vitellin: same double immunodiffusion.
B, vitellin Polyclonal Antibody Preparation: same double immunodiffusion.
The preparation of C, immune colloid gold:
1. the preparation of colloid gold particle: can obtain satisfied gold sol with trisodium citrate-tannic acid mixing reductive agent, working method is as follows: get the 4ml mass concentration and be 1% trisodium citrate (Na
3C
6H
5O
7.2H
2O), adding 5ml volumetric concentration is 1% tannic acid, 5ml, 25mmo/L K
2CO
2(volume equates with the tannic acid add-on), with bi-distilled water mend to the solution final volume be 20ml, be heated to 60 ℃ of HAuCl that get 1ml volumetric concentration 1%
4Be added in the 79ml bi-distilled water, heating in water bath to 60 ℃ adds above-mentioned citric acid-tan-liquor rapidly then, under this temperature, keep certain hour, after treating that solution colour becomes scarlet (needing 1 hour approximately), solution is heated to boiling, keeps boiling to get final product in 5 minutes.
2. proteinic processing: because the salt constituents can influence gold sol to absorption of proteins, and can make the colloidal sol coagulation, so should dialyse to the water of low ionic strength earlier before the sensitization.Must be noted that protein soln should definitely be clarified no fine particles, otherwise should remove with millipore filtration or ultracentrifugation earlier.Should avoid the existence of phosphate anion and borate ion generally speaking, because they all adsorbablely weaken Radioactive colloidal gold to absorption of proteins in particle surface.
3. the selection of protein optimum dose: after antibody dilution storage liquid to be marked made serial dilution, getting 0.1ml (containing protein 30 μ g) respectively is added in the 1ml colloidal gold solution, other establishes a pipe and does not add proteinic control tube, adding 0.1ml mass concentration is 10% NaCl solution after 5 minutes, left standstill behind the mixing 2 hours, coagulation will take place in unsettled gold sol, can make Radioactive colloidal gold the suitableeest stable protein content add 10% protein again and be the optimum mark protein content.
4. mark: use 0.1mol/L K
2CO
3Or 0.1mol/L HCl regulates colloidal gold solution pH=6.5; In the 100ml colloidal gold solution, add the antibody-solutions (volume is 3ml) of optimum mark amount, stirred 2～3 minutes; Adding 5ml volumetric concentration is 1% PEG20000 solution; In 12000rpm/min centrifugal 40 minutes, inhale and remove supernatant liquor (must guard against and topple over); Precipitation is suspended in certain volume contains in the damping fluid of 0.2～0.5mg/ml PEG20000, after the centrifugation, recover with same damping fluid again, concentration is advisable about with A 1cm/540nm=1.5, and is anticorrosion with the 0.5mg/ml sodium azide, puts 4 ℃ of preservations.
5. assembly method: the specking of film: Test line and Control line; The sealing of film; The processing of pad; The specking of gold marked reagent; To suck blood clearly with glass fibre, the anti-vitellin glass fibre of freeze-drying gold mark on plastic bottom board respectively, be fixed with the NC film of anti-vitellin antibody and hard absorbent filter by the figure assembling, accessory is bonding with the available double faced adhesive tape of combining of plastic bottom board or other cohesive material.The cardboard that assembles is cut into the strip that width is 4mm by longitudinal shear, test strip is encapsulated into aluminium foil bag or the plastic casing of packing into after sealing again, be and detect the male and female paper slip.
6. the result judges: what two pink vitta C and T band occurs is raun, and what a C band occurs is milter.
Claims (4)
1. the preparation method of sturgeon family fish ovovitellin antibody is characterized in that being prepared according to following step:
One, the preparation of sturgeon flying fish vitellin extracting solution:
1. take by weighing the Tris-HCl damping fluid of 0.02mol/l, the pH=8.0 of the fish-egg 5g of sturgeon section adding 15ml precooling in advance, high-speed homogenization, precipitation is abandoned in centrifugation, stays supernatant liquor;
2. supernatant liquor is by the gel chromatographic columns wash-out;
3. collect the elutriant that contains vitellin, the dialysis desalination obtains the vitellin extracting solution;
Two, sturgeon flying fish vitellin Polyclonal Antibody Preparation:
1. be lyophilized powder with the lyophilize of sturgeon vitellin extracting solution, the vitellin lyophilized powder that takes by weighing 1mg is dissolved in the 1ml physiological saline, adds the 1ml Freund's complete adjuvant, emulsification under the low temperature, and multi-point injection under the animal skin carries out initial immunity;
2. the 28th, 38, the 48 day subcutaneous multiple spot booster immunization in each back once, immunizing dose adopts Freund's incomplete adjuvant emulsification antigenic solution with for the first time;
3. after 55 days, animal ear edge extracts small amounts of blood and measures antibody titer;
4. the heart blood sampling prepares sturgeon flying fish vitellin antiserum(antisera).
2. according to the preparation method of the described sturgeon family fish ovovitellin antibody of claim 1, the filler that it is characterized in that gel chromatographic columns is sephadex G-100.
3. the application of the sturgeon family fish ovovitellin antibody of the described preparation method's preparation of claim 1 is characterized in that described sturgeon family fish ovovitellin antibody is used to differentiate sturgeon section fish male and female.
4. according to the application of the sturgeon family fish ovovitellin antibody of the described preparation method of claim 3 preparation, it is characterized in that differentiating that the method for sturgeon section fish male and female is double immunodiffusion, euzymelinked immunosorbent assay (ELISA) and immune colloid gold fast detection method.
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| KR102534027B1 (en) * | 2016-06-02 | 2023-05-18 | 호유 가부시키가이샤 | Egg allergy antigen |
| CN106053799A (en) * | 2016-08-03 | 2016-10-26 | 上海市水产研究所 | Gold-labeled test paper for rapid determination of crab vitellin content and application of colloidal-gold test paper |
| CN108168967A (en) * | 2017-12-12 | 2018-06-15 | 重庆师范大学 | A kind of preparation method of fish warning information element |
| CN109111515B (en) * | 2018-09-06 | 2021-05-14 | 大连工业大学 | Method for preparing main egg yolk protein from body cavity fluid of sea cucumber |
| CN109254156A (en) * | 2018-10-23 | 2019-01-22 | 山东出入境检验检疫局检验检疫技术中心 | Detect immune-blotting method kit and its application of Pseudopleuronectes fish vitellogenin |
| CN109374882A (en) * | 2018-10-23 | 2019-02-22 | 山东出入境检验检疫局检验检疫技术中心 | Detect Pseudopleuronectes fish vitellogenin immunity colloidal gold test paper strip and preparation method and application |
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