CN103833847A - Porcine circovirus-resistant refined egg yolk antibody and preparation method thereof - Google Patents
Porcine circovirus-resistant refined egg yolk antibody and preparation method thereof Download PDFInfo
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- CN103833847A CN103833847A CN201310651235.2A CN201310651235A CN103833847A CN 103833847 A CN103833847 A CN 103833847A CN 201310651235 A CN201310651235 A CN 201310651235A CN 103833847 A CN103833847 A CN 103833847A
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Abstract
The invention provides a porcine circovirus-resistant refined egg yolk antibody and a preparation method thereof. The preparation method comprises the following steps of adding an acetic acid buffer into immune egg yolk for dilution, adjusting a pH value to 4.8 by an acid, dropwisely adding octanoic acid into the dilute egg yolk solution with stirring at a room temperature in 30min according to a volume ratio of the dilute egg yolk solution to the octanoic acid of 1 milliliter to 11 microliters, carrying out standing at a low temperature for 2h, carrying out centrifugation at a rotation rate of 10000r/min for 30min, removing precipitates, filtering the supernatant by a nylon sieve of 125 micrometers, adding PBS of which the volume is 1/10 of that of the filtered supernatant and the content is 0.01mol/L into the filtered supernatant, adjusting a pH value to 7.2 by an alkali, mixing a formaldehyde solution having the content of 40% and the supernatant so that the final concentration of the formaldehyde solution is 0.1% (v/v), and carrying out stirring to obtain the desired product. The porcine circovirus-resistant refined egg yolk antibody can effectively prevent and treat porcine circovirus, is prepared by an octanoic acid precipitation method, has high purity, high titer and stable properties and can be preserved easily.
Description
Technical field
The present invention relates to biological technical field, especially a kind of resisting porcine circovirus refine yolk antibody and preparation method thereof.
Background technology
Pig circular ring virus is the minimum animal virus of one of finding so far.Known pig circular ring virus (PCV) has two serotypes, i.e. PCV-1 and PCV-2.Wherein, PCV-1 is non-virulent virus; PCV-2 is pathogenic virus; can cause pmws; pigskin inflammation and nephrotic syndrome, weanling pig and growing and fattening pigs prdc, the diseases such as children's congenital trembling of piglet in age (claiming again 2 type dependency central nervous system diseases); and can cause immunosuppression, bring very large difficulty to the control and prevention of disease of large-scale pig farm.
Mainly prevent the generation of this disease with vaccine immunity at present.Not for the specific medicament of virus disease, therefore on clinical treatment, mainly take the generation of symptomatic treatment and antibiotics complication prevention due to also, its result for the treatment of is undesirable.Therefore, be badly in need of aborning a kind of there is the good and curative effect of specificity, result for the treatment of for pig circular ring virus 2 and treating rapidly preparation.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of resisting porcine circovirus refine yolk antibody.
Another technical problem to be solved by this invention is to provide the preparation method of above-mentioned resisting porcine circovirus refine yolk antibody.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of resisting porcine circovirus refine yolk antibody, described antibody is obtained by following method:
(1) separate the high egg yolk of exempting from: the height that the immunity of porcine circovirus 2 type deactivation vaccine is crossed is exempted to send into the yellow and white separating machine after egg carries out disinfection and carried out yolk separation;
(2) prepare refine yolk antibody: in the yolk that step (1) is separated, add acetate buffer solution to dilute, wherein, the volume ratio of yolk and acetate buffer solution is 1:2, with acid tune PH to 4.8; It is sad dropwise to add under stirring at room temperature in every milliliter of dilution egg yolk liquid sad ratio that adds 11 μ L, in 30min, adds, and leaves standstill 2h under low temperature, then, with the centrifugal 30min of rotating speed of 10000r/min, abandons precipitation; Supernatant liquor, after 125 μ m nylon mesh are filtered, adds the PBS of the 0.01mol/L of 1/10 volume, with alkali tune PH to 7.2; 40% formaldehyde solution is mixed with this supernatant liquor, makes the final concentration of formaldehyde reach 0.1%(V/V), stir.
Preferably, above-mentioned resisting porcine circovirus refine yolk antibody, the antibody titer of described antibody is 1:128.
The preparation method of above-mentioned resisting porcine circovirus refine yolk antibody, concrete steps are as follows:
(1) separate the high egg yolk of exempting from: the height that the immunity of porcine circovirus 2 type deactivation vaccine is crossed is exempted to send into the yellow and white separating machine after egg carries out disinfection and carried out yolk separation;
(2) prepare refine yolk antibody: in the yolk that step (1) is separated, add acetate buffer solution to dilute, wherein, the volume ratio of yolk and acetate buffer solution is 1:2, with acid tune PH to 4.8; It is sad dropwise to add under stirring at room temperature in every milliliter of dilution egg yolk liquid sad ratio that adds 11 μ L, in 30min, adds, and leaves standstill 2h under low temperature, then, with the centrifugal 30min of rotating speed of 10000r/min, abandons precipitation; Supernatant liquor, after 125 μ m nylon mesh are filtered, adds the PBS of the 0.01mol/L of 1/10 volume, with alkali tune PH to 7.2; 40% formaldehyde solution is mixed with this supernatant liquor, makes the final concentration of formaldehyde reach 0.1%(V/V), stir;
(3) mensuration of antibody titer.
Preferably, the preparation method of above-mentioned resisting porcine circovirus refine yolk antibody, the sterilization method in described step (1) is height to be exempted to egg clear water rinse well, soak 3-5min with 0.05% hundred poisonings again, then dry with gauze, then clean one time with 75% ethanol, dry.
Preferably, the preparation method of above-mentioned resisting porcine circovirus refine yolk antibody, the height in described step (1) is exempted from egg and is obtained by following method:
(I) uses the high-yield egg chicken of porcine circovirus 2 type deactivation vaccine (commercially available) immune health;
(II) utilized immune high-yield egg chicken to obtain height and exempted from egg.
Preferably, the preparation method of above-mentioned resisting porcine circovirus refine yolk antibody, middle immune time >=2 with vaccine immunity high-yield egg chicken of described step (I).
Preferably, the preparation method of above-mentioned resisting porcine circovirus refine yolk antibody, the mensuration of described step (3) antibody titer refers to carries out pig circular ring virus antibody titer mensuration by final step (2) gained filtrate, after aseptic PBS doubling dilution with 0.01M, pH7.2, add agar gel around in hole, in interstitial hole, add pig circular ring virus antigen, put 37 ℃ of incubators reaction 24h in wet box, take the egg yolk liquid of high dilution that occurs sealed Belt as the antibody titer of the pig circular ring virus of this egg yolk liquid.
The invention has the beneficial effects as follows:
Above-mentioned resisting porcine circovirus refine yolk antibody, is a kind of have the good and curative effect of specificity, result for the treatment of for pig annulus disease and treat rapidly preparation, can carry out effectively preventing for pig circular ring virus 2; Its preparation method adopts the sad precipitator method to extract resisting porcine circovirus refine yolk antibody, and this method is safe, cheap, is suitable for scale operation, and the yolk antibody purity of preparation is high, the height of tiring, stable in properties, be easy to preserve.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is further described.
Experimental technique in following embodiment, if no special instructions, is ordinary method; Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Embodiment 1
Prepare the height of pig circular ring virus and exempt from egg
(1) select the not immune high-yield egg chicken isolated rearing of health, strengthen feed nutrition, add hello ceftiofur sodium simultaneously and infect with pre-bacteriological protection;
(2) high-yield egg chicken of processing by commercially available porcine circovirus 2 type deactivation vaccine immunity step (1), concrete steps are: carry out three immunity, head exempts from posterior auricular muscle meat injection 1-2ml, after 2 weeks, two exempt from, injecting immune vaccine 1-2ml, then exempt from injecting immune vaccine 1-2ml every 2 Wednesdays;
(3) after immune high-yield egg chicken, collect the height obtaining and exempt from egg, be the high egg of exempting from of pig circular ring virus.
Embodiment 2
The high egg of exempting from of pig circular ring virus that utilizes embodiment 1 to prepare is prepared refine yolk antibody
(1) separate the high egg yolk of exempting from:
By height described in embodiment (1) exempt from egg two exempt from after 1 week start every 7 days sampling measure high exempt from egg yolk in resisting porcine circovirus specific antibody tire, antibody titer all reaches 1:128 and collects yolk when above, the concrete grammar of described collection yolk is: the height that embodiment 1 is obtained is exempted from egg clear water and cleaned, soak 3-5min with 0.05% hundred poisonings again, then dry with gauze, clean one time with 75% ethanol again, dry, send into the yellow and white separating machine and carry out yolk separation;
(2) prepare refine yolk antibody:
In the yolk that step (1) is separated, add acetate buffer solution to dilute, wherein, the volume ratio of yolk and acetate buffer solution is 1:2, described acetate buffer solution refers to the acetate buffer of PH3.5, get ammonium acetate 25g, the 25ml that adds water adds 7mol/L hydrochloric acid soln 38ml after dissolving, accurately regulate pH value to 3.5(potentiometric titration with 2mol/L hydrochloric acid soln or 5mol/L ammonia solution), be diluted with water to 100ml and get final product; With acid tune PH to 4.8; It is sad dropwise to add under stirring at room temperature in every milliliter of dilution egg yolk liquid sad ratio that adds 11 μ L, in 30min, adds, and leaves standstill 2h under low temperature, then, with the centrifugal 30min of rotating speed of 10000r/min, abandons precipitation; Supernatant liquor, after 125 μ m nylon mesh are filtered, adds the PBS of the 0.01mol/L of 1/10 volume, with alkali tune PH to 7.2; 40% formaldehyde solution is mixed with this supernatant liquor, makes the final concentration of formaldehyde reach 0.1%(V/V), stir;
(3) mensuration of antibody titer:
Final step (2) gained filtrate is carried out to pig circular ring virus antibody titer mensuration, after aseptic PBS doubling dilution with 0.01M, pH7.2, add agar gel around in hole, in interstitial hole, add pig circular ring virus antigen, put 37 ℃ of incubators reaction 24h in wet box, take the egg yolk liquid of high dilution that occurs sealed Belt as the antibody titer of the pig circular ring virus of this egg yolk liquid.
Application example
The clinical effect trial of resisting porcine circovirus refine yolk antibody to pig annulus disease
1, materials and methods
(1) test materials
Utilize the resisting porcine circovirus refine yolk antibody that in embodiment 2, method prepares, antibody titer is 1:128.
The strong malicious strain isolated of strain pig circular ring virus.
30 of laboratory animal 35 age in days sodium selenites.
(2) test method
30 of the sodium selenites that selection is pig circular ring virus negative antibody through pig circular ring virus antibody ELISA diagnosis reagent kit (purchased from Shenzhen Lv Shiyuan Bioisystech Co., Ltd) detection, isolated rearing was observed after one week, was for experiment.
Test pig is divided into three groups at random, and first group is blank group (10), and second group for attacking malicious control group (10), and the 3rd group for attacking malicious treatment group (10).First group is blank group, does not inject any medicine, isolated rearing.Second group and the 3rd group of strong poison of every intramuscular injection pig circular ring virus (TCID50:106.5/ml) 2ml.In attack latter 24 hours of poison to pig circular ring virus refine yolk antibody described in the 3rd group of every scalp hemostasis 2ml embodiment 2 (fine jade expands and tires: 1:128), second group of subcutaneous injection physiological saline 2ml.The morbidity of observation pig and death condition to the 21 days.
2, test results and analysis
The clinical effect trial result of resisting porcine circovirus refine yolk antibody to pig annulus disease, refers to table 1.
The clinical effect trial result of table 1 resisting porcine circovirus refine yolk antibody to pig annulus disease
| Numbering | Group | Sickness rate % | Mortality ratio % | Efficient % |
| First group | Blank group | 0 | 0 | 100 |
| Second group | Attack malicious control group | 90 | 80 | 20 |
| The 3rd group | Attack malicious treatment group | 30 | 0 | 100 |
From table 1, first group there is not any clinical symptom, not morbidity and dead; Second group has 9 hair diseases after attacking poison, 8 death, and sick pig shows as fervescence more than 41 ° in latter 2 days in attacking poison, expiratory dyspnea, appetite declines, and enjoys and the symptom such as diarrhoea; The 3rd group has 3 hair diseases, there is no death after attacking poison.Sick pig shows as the symptoms such as appetite declines, and spirit is depressed, slight cough for latter 3 days in attacking poison, but after 5 days, symptom alleviates rear recovery gradually.
Test-results shows: resisting porcine circovirus refine yolk antibody has good therapeutic action to pig annulus disease, evident in efficacy.
Above-mentioned detailed description of this kind of resisting porcine circovirus refine yolk antibody and preparation method thereof being carried out with reference to embodiment; illustrative rather than determinate; can list several embodiment according to institute's limited range; therefore in the variation and the modification that do not depart under general plotting of the present invention, within should belonging to protection scope of the present invention.
Claims (7)
1. a resisting porcine circovirus refine yolk antibody, is characterized in that: described antibody is obtained by following method:
(1) separate the high egg yolk of exempting from: the height that the immunity of porcine circovirus 2 type deactivation vaccine is crossed is exempted to send into the yellow and white separating machine after egg carries out disinfection and carried out yolk separation;
(2) prepare refine yolk antibody: in the yolk that step (1) is separated, add acetate buffer solution to dilute, wherein, the volume ratio of yolk and acetate buffer solution is 1:2, with acid tune PH to 4.8; It is sad dropwise to add under stirring at room temperature in every milliliter of dilution egg yolk liquid sad ratio that adds 11 μ L, in 30min, adds, and leaves standstill 2h under low temperature, then, with the centrifugal 30min of rotating speed of 10000r/min, abandons precipitation; Supernatant liquor, after 125 μ m nylon mesh are filtered, adds the PBS of the 0.01mol/L of 1/10 volume, with alkali tune PH to 7.2; 40% formaldehyde solution is mixed with this supernatant liquor, makes the final concentration of formaldehyde reach 0.1%(V/V), stir.
2. resisting porcine circovirus refine yolk antibody according to claim 1, is characterized in that: the antibody titer of described antibody is 1:128.
3. the preparation method of resisting porcine circovirus refine yolk antibody described in claim 1, is characterized in that: concrete steps are as follows:
(1) separate the high egg yolk of exempting from: the height that the immunity of porcine circovirus 2 type deactivation vaccine is crossed is exempted to send into the yellow and white separating machine after egg carries out disinfection and carried out yolk separation;
(2) prepare refine yolk antibody: in the yolk that step (1) is separated, add acetate buffer solution to dilute, wherein, the volume ratio of yolk and acetate buffer solution is 1:2, with acid tune PH to 4.8; It is sad dropwise to add under stirring at room temperature in every milliliter of dilution egg yolk liquid sad ratio that adds 11 μ L, in 30min, adds, and leaves standstill 2h under low temperature, then, with the centrifugal 30min of rotating speed of 10000r/min, abandons precipitation; Supernatant liquor, after 125 μ m nylon mesh are filtered, adds the PBS of the 0.01mol/L of 1/10 volume, with alkali tune PH to 7.2; 40% formaldehyde solution is mixed with this supernatant liquor, makes the final concentration of formaldehyde reach 0.1%(V/V), stir;
(3) mensuration of antibody titer.
4. the preparation method of resisting porcine circovirus refine yolk antibody according to claim 3, it is characterized in that: the sterilization method in described step (1) is height to be exempted to egg clear water rinse well, soak 3-5min with 0.05% hundred poisonings again, then dry with gauze, clean one time with 75% ethanol again, dry.
5. the preparation method of resisting porcine circovirus refine yolk antibody according to claim 3, is characterized in that: the height in described step (1) is exempted from egg and obtained by following method:
(I) uses the high-yield egg chicken of porcine circovirus 2 type deactivation vaccine immune health;
(II) utilized immune high-yield egg chicken to obtain height and exempted from egg.
6. the preparation method of resisting porcine circovirus refine yolk antibody according to claim 5, is characterized in that: immune time >=2 of using vaccine immunity high-yield egg chicken in described step (I).
7. the preparation method of resisting porcine circovirus refine yolk antibody according to claim 3, it is characterized in that: the mensuration of described step (3) antibody titer refers to carries out pig circular ring virus antibody titer mensuration by final step (2) gained filtrate, after aseptic PBS doubling dilution with 0.01M, pH7.2, add agar gel around in hole, in interstitial hole, add pig circular ring virus antigen, put 37 ℃ of incubators reaction 24h in wet box, take the egg yolk liquid of high dilution that occurs sealed Belt as the antibody titer of the pig circular ring virus of this egg yolk liquid.
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| CN102617731A (en) * | 2012-04-19 | 2012-08-01 | 华中农业大学 | Porcine circovirus-resistant type 2 egg yolk antibody and preparation method and application thereof |
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| CN102617731A (en) * | 2012-04-19 | 2012-08-01 | 华中农业大学 | Porcine circovirus-resistant type 2 egg yolk antibody and preparation method and application thereof |
| CN102872460A (en) * | 2012-09-29 | 2013-01-16 | 天津市中升挑战生物工程有限公司 | Refined egg yolk antibody compound for preventing and treating duck reovirus diseases and method for preparing refined egg yolk antibody compound |
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| CN104311661A (en) * | 2014-10-17 | 2015-01-28 | 挑战（天津）动物药业有限公司 | Preparation method of anti-escherichia coli quadruple yolk antibody |
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Application publication date: 20140604 |