CN101575374B - Chicken Marek's virus antibody and preparation method thereof - Google Patents
Chicken Marek's virus antibody and preparation method thereof Download PDFInfo
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- CN101575374B CN101575374B CN 200910027855 CN200910027855A CN101575374B CN 101575374 B CN101575374 B CN 101575374B CN 200910027855 CN200910027855 CN 200910027855 CN 200910027855 A CN200910027855 A CN 200910027855A CN 101575374 B CN101575374 B CN 101575374B
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Abstract
The invention discloses a chicken Marek's virus antibody and a preparation method thereof. The preparation method comprises the following steps: adopting at least one of Marek's disease (MD) virus (MDV) vaccine strain, expression vector containing an objective antibody, pathogenic Marek's virus and MDV antigen adjuvant vaccine to inoculate a chicken according to a certain inoculation program, monitoring the objective antibody potency generated by the inoculated laying chicken, and beginning collecting eggs produced by the inoculated laying chicken when the prescribed lowest objective antibody potency is achieved; and separating egg yolks in the collected eggs, preparing egg yolk supernatant by adopting an acid water method, ultra-filtering the egg yolk supernatant to obtain concentrated solution, and carrying out degreasing and purification treatment for the concentrated solution by adopting chloroform to obtain the objective antibody. Therefore, on the basis of not influencing the specificity and sensibility of the antibody, the preparation of the antibody is safer, simpler, more convenient and feasible, and is convenient for industrialization; and the prepared antibody has high yield and low cost, and can be used for immune monitoring and plague prevention and control.
Description
Technical field
The present invention relates to a kind of biotechnological formulation and preparation method thereof, particularly relate to a kind of chicken Marek's virus antibody and preparation method thereof.
Background technology
Marek (MD) is inhibitive ability of immunity and the tumorigenicity transmissible disease that is caused by Mareks simplexvirus (MDV), and its latent period is long, and is disguised strong, is one of Infectious Diseases of chicken.Because this disease grievous injury chicken immune system, except itself causing accumulative total up to the lethality rate more than 70%, also often can cause the immuning failure of other chicken disease vaccine or secondary infection and cause heavy losses.The MD vaccine immunity only can stop or reduce chicken infected MD morbidity, and can not block pathogenic MDV to chicken group's intrusion and propagation.Therefore, the important means that MD prevents and treats is not only in diagnosis and the monitoring of MDV infection, also is prerequisite and the effective ways of estimating foster fowl Biosafety situation that conclusive evidence fowl is used immune effect of vaccine.
MDV antigen is the meta-bolites that MDV produces in breeding.Utilize immunoprecipitation method can from the cells infected extract of MDV GA strain, identify nearly 46 kinds of virus-specific polypeptide, wherein, can identify 3 main MDV antigens by agar diffusion test, difference called after Staphylococal Protein A, B antigen and C antigen, these viral antigens can excite infection immunity chicken body to produce corresponding antibody.
MDV has three serotypes, and very strong tumorigenesis strain and their weakening strain all are classified as serum I type MDV from the mild to the virulence, and the virus strain of natural not tumorigenesis is the serum II type, and herpes turkey virus (HVT) belongs to serum II I type.Only having the virulent strain in the serum I type in three serotypes is tumorigenesis or pathogenic to chicken, and the non-tumorigenesis strain of three serotypes all can be used as the vaccine of MD.Wherein, it is significantly different that the agp antigen that MDV serum II type vaccine produces and MDV Staphylococal Protein A have, and the HVT vaccine produces Staphylococal Protein A in immune chicken body and institute's corresponding antibody that excites is all very limited, and the classical agar diffusion test of muting sensitive is difficult to detect.
At present, preparation MDV positive antibody, to separate the serum of immunity survival chicken behind pathogenic MDV infected chicken, immunoinfective or MDV antigen immune chicken to produce mostly, therefore, the method of this MDV positive antibody preparation exists the danger of loose poison all the time, test chicken group's raising, management etc. must be taked strict Measures of Biosafety Control, from and cause the production cost of positive serum very high; Simultaneously, in the positive serum of preparation, contain a large amount of non-purpose antibody and the potential cause of disease that may exist, thereby be difficult to be allowed to enter regular production channel.
At present, the method for extracting both at home and abroad antibody from egg yolk has multiple, and wherein relevant with present patent application have two kinds.A kind of method comprises following processing step: (1) directly adds a large amount of chloroforms in yolk liquid, with its aqueous solution of extracting; (2) adopt the further aqueous solution of concentrated chloroform extracting of hyperfiltration process, can obtain the purpose antibody in the yolk.Another kind method comprises following processing step: (1) preparation yolk supernatant liquor; (2) adopt chloroform that the yolk supernatant liquor is carried out the degreasing purification process; (3) carry out ultrafiltration and obtain concentrated solution, can obtain the purpose antibody in the yolk.All there is following problem in above-mentioned two kinds of methods of extracting yolk antibody: owing to adopt chloroform directly yolk liquid or yolk supernatant liquor to be carried out the degreasing purification process first, carry out ultrafiltration, therefore, the consumption of chloroform is many again, the workload of purifying is large, and the preparation cost of yolk antibody is high.
Summary of the invention
Technical problem to be solved by this invention provides a kind of chicken Marek's virus antibody and preparation method thereof, like this, on the basis of the specificity that does not affect antibody and susceptibility, can make the preparation of antibody safer, simple and easy to do and be convenient to industrialization, and the antibody production that makes is high, cost is low, can be used for immunologic surveillance and control and prevention of disease.
For solving the problems of the technologies described above, the invention provides a kind of chicken Marek's virus antibody, this antibody makes by following steps:
(1) adopts at least a in Marek (MD) virus (MDV) vaccine strain, the expression vector that contains purpose antigen, pathogenic Mareks disease virus and the MDV antigen adjuvant vaccine, by certain vaccine program inoculation chicken, the purpose antibody titer that monitoring inoculation laying hen produces, and when it reaches the minimum purpose antibody titer of regulation, begin to collect the egg that this inoculation laying hen produces;
(2) isolate yolk in the egg of collection, adopt the standby yolk supernatant liquor of sour water legal system, the yolk supernatant liquor is carried out ultrafiltration and obtains concentrated solution, adopt again chloroform that concentrated solution is carried out the degreasing purification process, obtain purpose antibody.
The present invention uses can produce or discharge the biotechnological formulation of purpose Mareks disease virus antigen as immunogen, adopt certain program inoculation chicken, utilize the inoculation laying hen as bio-reactor production specific biological active substance-antibody, this antibody can enter yolk in the egg by blood, by to after the detection of this antibody and reaching the tiring of regulation, can collect inoculation egg that laying hen produces, and take certain treatment process to extract yolk antibody.
The production that the present invention uses yolk antibody comes the production of alternative serum antibody, an egg the antibody amount that obtains can surpass the serum antibody amount of a bloodletting gained of conventional infected chicken, greatly reduce the production cost of purpose antibody, and reduced to greatest extent the pathogen contamination in the production process.In the extraction of yolk antibody, adopt first the sour water method slightly to propose the yolk supernatant liquor to the egg of the inoculation laying hen of collecting, the yolk supernatant liquor is carried out ultrafiltration and obtains concentrated solution, adopt again chloroform that concentrated solution is carried out the degreasing purification process, obtain purpose antibody.Like this, the workload of chloroform degreasing purification process can be significantly reduced, and the chloroform consumption can be saved more than 50%.Therefore, the present invention prepares purpose antibody by the egg that the inoculation laying hen produces, on the basis of the specificity that does not affect antibody and susceptibility, can make the preparation of antibody safer, simple and easy to do and be convenient to industrialization, and the antibody production that makes is high, cost is low, can be used for immunologic surveillance and control and prevention of disease.
In preparation method of the present invention:
The step of described inoculation chicken is: healthy chick's inoculation Marek (MD) virus (MDV) vaccine strain is as fundamental immunity, interval 0.5～1.5 month, with a kind of booster immunization that carries out in the expression vector that contains purpose antigen, pathogenic Mareks disease virus and the MDV antigen adjuvant vaccine, after, carried out reinforced immunological every 2～3 months with MDV antigen adjuvant vaccine.
The step of described inoculation chicken is: the laying hen inoculation MDV antigen adjuvant vaccine that 3 monthly ages are above or the expression vector that contains purpose antigen are as fundamental immunity, interval 1～1.5 month, carry out booster immunization with MDV antigen adjuvant vaccine, after, carried out reinforced immunological every 2～3 months with MDV antigen adjuvant vaccine.
The process of the standby yolk supernatant liquor of described employing sour water legal system is: with yolk, Acetate-acetate buffer solution in 1: the ratio of (6～14) mixes, filtered through gauze is to remove vitelline membrane, and be statically placed in 6～24h under the room temperature, make the yolk supernatant liquor, the pH value of described Acetate-acetate buffer solution is 4.5～5.5, concentration is 0.05～0.12mol/L.
It is the ultra-filtration membrane of 10～150KD that molecular weight cut-off is adopted in the described ultrafiltration that the yolk supernatant liquor is carried out, and operation pressure reduction is 0.01～0.2Mpa, and the concentrated solution volume that obtains is 1/5～1/2 of yolk volume.
Described employing chloroform to the process that concentrated solution carries out the degreasing purification process is: after adding final concentration and be 1～5% chloroform in the yolk supernatant liquor being carried out the concentrated solution that ultrafiltration obtains, concussion 3～5min, under 4 ℃ temperature condition, leave standstill 30min～72h, use again the centrifugal 10～60min of centrifugal force of 8000～10000 * g, collect supernatant liquor, and to regulate its pH value be 6.0～7.4, namely obtains the purpose antibody of clear.
Described Marek (MD) virus (MDV) vaccine strain comprises the CVI988 strain of serum I type, the herpes turkey virus Fc-126 strain of serum II I type; The serum I type MDV gentle strain of described pathogenic Mareks disease virus for can effectively secreting Mareks disease virus purpose antigen.
The preparation method of described MDV antigen adjuvant vaccine is: the MDV antigen that will extract purifying carries out emulsification preparation and check with oil phase after being made into according to a conventional method water, and MDV antigen comprises MDV Staphylococal Protein A, MDV B antigen.
When collecting egg, the fine jade that reaches the purpose antibody of regulation in the egg expands to tire and is not less than 1: 4, or with tiring that other method that described fine jade expansion is tired suitable is surveyed.
In addition, described Marek (MD) virus (MDV) vaccine strain is not only limited to the herpes turkey virus living vaccine of serum I type living vaccine, serum II I type, can be other Mareks disease virus commercially available vaccine that can effectively secrete Mareks disease virus purpose antigen yet.
In described MDV antigen adjuvant vaccine, also can add immunopotentiating agent, or in chicken inoculation MDV antigen adjuvant vaccine, use in addition immunopotentiating agent.
As of the present invention preferred, described pathogenic Mareks disease virus is the BJ-1 strain.
Embodiment
Embodiment one:
A kind of preparation method of chicken Marek's virus antibody may further comprise the steps:
(1) select newborn SPF healthy chick, inoculation Marek (MD) virus (MDV) vaccine strain is as fundamental immunity, and described vaccine strain is that CVI988 strain (commercially available) and the by specification of serum I type requires to carry out immunity;
Immunity is carried out booster immunization with MDV antigen adjuvant vaccine by the subcutaneous injection of 0.5ml/ plumage dosage in the time of rear 30 days, the preparation method of described Adjuvanted vaccines is: the MDV Staphylococal Protein A that will extract purifying carries out the emulsification preparation with oil phase after being made into according to a conventional method water, requires fine jade to expand and tires more than 1: 8;
Carry out reinforced immunological with this MDV antigen adjuvant vaccine by the intramuscular injection of 1.0ml/ plumage dosage again the 60th day the time behind the booster immunization, carry out once identical reinforced immunological with this MDV antigen adjuvant vaccine more at quarterly intervals later on;
Behind the inoculation Egg Production of Laying Hens, begin to monitor the inoculation laying hen and produce purpose antibody titer level in the egg, tire when being not less than 1: 4 when the fine jade expansion of the purpose antibody that reaches regulation in the egg, begin to collect the egg that this inoculation laying hen produces;
(2) isolate yolk in the egg of collection, adopt the standby yolk supernatant liquor of sour water legal system: yolk, Acetate-acetate buffer solution are mixed in 1: 6 ratio, filtered through gauze is to remove vitelline membrane, and be statically placed in 24h under the room temperature, make the yolk supernatant liquor, the pH value of described Acetate-acetate buffer solution is 5.5, concentration is 0.12mol/L;
The yolk supernatant liquor is carried out ultrafiltration and obtains concentrated solution: it is the ultra-filtration membrane of 150KD that molecular weight cut-off is adopted in ultrafiltration, and operation pressure reduction is 0.01Mpa, and the concentrated solution volume that obtains is 1/5 of yolk volume;
Adopt chloroform that concentrated solution is carried out the degreasing purification process: after in the yolk supernatant liquor being carried out the concentrated solution that ultrafiltration obtains, adding final concentration and be 1.5% chloroform, concussion 5min, under 4 ℃ temperature condition, leave standstill 30min, use again the centrifugal 10min of centrifugal force of 10000 * g, collect supernatant liquor, and to regulate its pH value be 7.0, namely obtains the purpose antibody of clear.
Embodiment two:
A kind of preparation method of chicken Marek's virus antibody may further comprise the steps:
(1) select newborn SPF healthy chick, inoculation Marek (MD) virus (MDV) vaccine strain is as fundamental immunity, and described vaccine strain is that CVI988 strain (commercially available) and the by specification of serum I type requires to carry out immunity;
Immunity is rear 45 days the time, the poison of the blood after the BJ-1 strain rejuvenation of pathogenic Mareks disease virus is carried out 100 times of dilutions with MD vaccine special use diluent after, carry out booster immunization by the subcutaneous injection of 0.2ml/ plumage dosage;
Carry out reinforced immunological with MDV antigen adjuvant vaccine by the intramuscular injection of 1.5ml/ plumage dosage again 60 days the time behind the booster immunization, later carried out once identical reinforced immunological every 2 months with this MDV antigen adjuvant vaccine, the preparation method of described Adjuvanted vaccines is: the MDV Staphylococal Protein A that will extract purifying carries out the emulsification preparation with oil phase after being made into according to a conventional method water, requires fine jade to expand and tires more than 1: 8;
Behind the inoculation Egg Production of Laying Hens, begin to monitor the inoculation laying hen and produce purpose antibody titer level in the egg, tire when being not less than 1: 4 when the fine jade expansion of the purpose antibody that reaches regulation in the egg, begin to collect the egg that this inoculation laying hen produces;
(2) isolate yolk in the egg of collection, adopt the standby yolk supernatant liquor of sour water legal system: yolk, Acetate-acetate buffer solution are mixed in 1: 12 ratio, filtered through gauze is to remove vitelline membrane, and be statically placed in 12h under the room temperature, make the yolk supernatant liquor, the pH value of described Acetate-acetate buffer solution is 4.5, concentration is 0.05mol/L;
The yolk supernatant liquor is carried out ultrafiltration and obtains concentrated solution: it is the ultra-filtration membrane of 10KD that molecular weight cut-off is adopted in ultrafiltration, and operation pressure reduction is 0.2Mpa, and the concentrated solution volume that obtains is 1/2 of yolk volume;
Adopt chloroform that concentrated solution is carried out the degreasing purification process: after in the yolk supernatant liquor being carried out the concentrated solution that ultrafiltration obtains, adding final concentration and be 1.0% chloroform, concussion 3min, under 4 ℃ temperature condition, leave standstill 12h, use again the centrifugal 10min of centrifugal force of 8000 * g, collect supernatant liquor, and to regulate its pH value be 6.0, namely obtains the purpose antibody of clear.
Embodiment three:
A kind of preparation method of chicken Marek's virus antibody may further comprise the steps:
(1) require immune newborn SPF healthy chick with herpes turkey virus Fc-126 strain (commercially available) by specification of the serum II I type of Marek (MD) virus (MDV) vaccine strain, immunity in the time of rear 15 days again CVI988 strain (commercially available) by specification with the serum I type of Marek (MD) virus (MDV) vaccine strain require to carry out second immunisation as fundamental immunity;
After the fundamental immunity 15 days the time, carry out booster immunization with MDV antigen adjuvant vaccine by the intramuscular injection of 0.5ml/ plumage dosage, the preparation method of described Adjuvanted vaccines is: the MDV Staphylococal Protein A that will extract purifying carries out the emulsification preparation with oil phase after being made into according to a conventional method water, requires fine jade to expand and tires more than 1: 16;
Carry out reinforced immunological with this MDV antigen adjuvant vaccine by the intramuscular injection of 1.0ml/ plumage dosage again 60 days the time behind the booster immunization, after this carried out once identical reinforced immunological every 2 months with MDV antigen adjuvant vaccine;
The fine jade that produces purpose antibody in the egg when this inoculation chicken expands to tire and reaches 1: 4 and collect the egg that this inoculation laying hen produces when above;
(2) isolate yolk in the egg of collection, adopt the standby yolk supernatant liquor of sour water legal system: yolk, Acetate-acetate buffer solution are mixed in 1: 14 ratio, filtered through gauze is to remove vitelline membrane, and be statically placed in 6h under the room temperature, make the yolk supernatant liquor, the pH value of described Acetate-acetate buffer solution is 5.0, concentration is 0.10mol/L;
The yolk supernatant liquor is carried out ultrafiltration and obtains concentrated solution: it is the ultra-filtration membrane of 100KD that molecular weight cut-off is adopted in ultrafiltration, and operation pressure reduction is 0.1Mpa, and the concentrated solution volume that obtains is 3/10 of yolk volume;
Adopt chloroform that concentrated solution is carried out the degreasing purifying: after in the yolk supernatant liquor being carried out the concentrated solution that ultrafiltration obtains, adding final concentration and be 5% chloroform, concussion 5min, under 4 ℃ temperature condition, leave standstill 72h, use again the centrifugal 60min of centrifugal force of 9000 * g, collect supernatant liquor, and to regulate its pH value be 7.4, namely obtains the purpose antibody of clear.
Embodiment four:
A kind of preparation method of chicken Marek's virus antibody may further comprise the steps:
(1) selects newborn SPF healthy chick, inoculation Marek (MD) virus (MDV) vaccine strain is as fundamental immunity, and described vaccine strain is that herpes turkey virus Fc-126 strain (commercially available) and the by specification of serum II I type requires to carry out immunity;
After the fundamental immunity 40 days the time, the poison of the blood after the BJ-1 strain rejuvenation of pathogenic Mareks disease virus carried out 100 times of dilutions with MD vaccine special use diluent after, carry out booster immunization by the subcutaneous injection of 0.2ml/ plumage dosage;
Carry out reinforced immunological with MDV antigen adjuvant vaccine by the intramuscular injection of 1.0ml/ plumage dosage 60 days the time behind the booster immunization, after this carry out once identical reinforced immunological with this MDV antigen adjuvant vaccine at quarterly intervals, the preparation of described MDV antigen adjuvant vaccine is with embodiment three;
Behind the inoculation Egg Production of Laying Hens, begin to monitor the inoculation laying hen and produce purpose antibody titer level in the egg, tire when being not less than 1: 4 when the fine jade expansion of the purpose antibody that reaches regulation in the egg, begin to collect the egg that this inoculation laying hen produces;
(2) thereafter the preparation of chicken Marek's virus antibody is with embodiment one.
Embodiment five:
A kind of preparation method of chicken Marek's virus antibody may further comprise the steps:
(1) selects the SPF laying hen at 3 monthly ages, with MDV antigen adjuvant vaccine as immunogen, press the intramuscular injection of 0.5ml/ plumage dosage as fundamental immunity, the preparation method of described MDV antigen adjuvant vaccine is: the MDV Staphylococal Protein A that will extract purifying carries out the emulsification preparation with oil phase after being made into according to a conventional method water, requires fine jade to expand and tires more than 1: 16;
After fundamental immunity, carry out booster immunization with this MDV antigen adjuvant vaccine by the intramuscular injection of 1.0ml/ plumage dosage 30 days the time;
Carry out reinforced immunological with this MDV antigen adjuvant vaccine by the intramuscular injection of 1.5ml/ plumage dosage again 70 days the time behind the booster immunization, after this, carried out once identical reinforced immunological every 2 months with this MDV antigen adjuvant vaccine;
Monitoring inoculation laying hen produces the purpose antibody titer level in the egg, tires when being not less than 1: 4 when the fine jade expansion of the purpose antibody that reaches regulation in the egg, begins to collect the egg that this inoculation laying hen produces.
(2) thereafter the preparation of chicken Marek's virus antibody is with embodiment two.
Embodiment six:
A kind of preparation method of chicken Marek's virus antibody may further comprise the steps:
(1) select newborn SPF healthy chick, inoculation Marek (MD) virus (MDV) vaccine strain is as fundamental immunity, and described vaccine strain is that CVI988 strain (commercially available) and the by specification of serum I type requires to carry out immunity;
Immunity is carried out booster immunization with the expression vector that contains purpose antigen by 150 μ g/ plumage dosage intramuscular injection in the time of rear 30 days;
Carry out reinforced immunological with MDV antigen adjuvant vaccine by the intramuscular injection of 1.0ml/ plumage dosage again 2 months the time behind the booster immunization, the preparation method of described MDV antigen adjuvant vaccine carried out once identical reinforced immunological every 2.5 months with same MDV antigen adjuvant vaccine later with embodiment one;
Behind the inoculation Egg Production of Laying Hens, begin to monitor the inoculation laying hen and produce purpose antibody titer level in the egg, tire when being not less than 1: 4 when the fine jade expansion of the purpose antibody that reaches regulation in the egg, begin to collect the egg that this inoculation laying hen produces;
(2) thereafter the preparation of chicken Marek's virus antibody is with embodiment one.
Embodiment seven:
A kind of preparation method of chicken Marek's virus antibody may further comprise the steps:
(1) select the SPF laying hen at 3 monthly ages, with the expression vector that contains purpose antigen by 200 μ g/ plumage dosage intramuscular injection as fundamental immunity;
After fundamental immunity 45 days, carry out subcutaneous injection as booster immunization with MDV antigen adjuvant vaccine by 1.0ml/ plumage dosage, the preparation method of described MDV antigen adjuvant vaccine is with embodiment three;
Behind booster immunization, carry out reinforced immunological with this MDV antigen adjuvant vaccine by the intramuscular injection of 1.5ml/ plumage dosage again in 60 days, after this, carry out once identical reinforced immunological with this MDV antigen adjuvant vaccine at quarterly intervals;
Monitoring inoculation laying hen produces the purpose antibody titer level in the egg, tires when being not less than 1: 4 when the fine jade expansion of the purpose antibody that reaches regulation in the egg, begins to collect the egg that this inoculation laying hen produces.
(2) thereafter the preparation of chicken Marek's virus antibody is with embodiment three.
Above-described only is seven kinds of embodiments of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the principle of the invention, can also make some modification and improvement, these all should be considered as belonging to protection scope of the present invention.Such as: MDV antigen also can be MDV B antigen; Making lyophilized antibodies with the purpose antibody of the liquid phase that makes is beneficial to preserve and use; When antibody titer keeps higher level or drops to lower level, can delay or implement in advance reinforced immunological; Because of the difference of manufacturer, selected ultra-filtration membrane should be lived antibody with effectively catching and is as the criterion, and can not only be as the criterion according to the molecular weight cut-off that marks.
Claims (8)
1. the preparation method of a chicken Marek's virus antibody may further comprise the steps:
(1) adopts at least a in Marek's disease virus vaccine strain, the expression vector that contains purpose antigen, pathogenic Mareks disease virus and the MDV antigen adjuvant vaccine, by certain vaccine program inoculation chicken, the fine jade of the purpose antibody titer that monitoring inoculation laying hen produces expands tires when being not less than 1:4, begins to collect the egg that this inoculation laying hen produces;
The step of described inoculation chicken is: healthy chick's inoculation Marek's disease virus vaccine strain is as fundamental immunity, interval 0.5～1.5 month, with a kind of booster immunization that carries out in the expression vector that contains purpose antigen, pathogenic Mareks disease virus and the MDV antigen adjuvant vaccine, after, carried out reinforced immunological every 2～3 months with MDV antigen adjuvant vaccine;
(2) isolate yolk in the egg of collection, adopt the standby yolk supernatant liquor of sour water legal system, the yolk supernatant liquor is carried out ultrafiltration and obtains concentrated solution, adopt again chloroform that concentrated solution is carried out the degreasing purification process, obtain purpose antibody.
2. the preparation method of a chicken Marek's virus antibody may further comprise the steps:
(1) adopts at least a in the expression vector contain purpose antigen, the MDV antigen adjuvant vaccine, by certain vaccine program inoculation chicken, the fine jade of the purpose antibody titer that monitoring inoculation laying hen produces expands tires when being not less than 1:4, begins to collect the egg that this inoculation laying hen produces;
The step of described inoculation chicken is: the laying hen inoculation MDV antigen adjuvant vaccine that 3 monthly ages are above or the expression vector that contains purpose antigen are as fundamental immunity, interval 1～1.5 month, carry out booster immunization with MDV antigen adjuvant vaccine, after, carried out reinforced immunological every 2～3 months with MDV antigen adjuvant vaccine;
(2) isolate yolk in the egg of collection, adopt the standby yolk supernatant liquor of sour water legal system, the yolk supernatant liquor is carried out ultrafiltration and obtains concentrated solution, adopt again chloroform that concentrated solution is carried out the degreasing purification process, obtain purpose antibody.
3. the preparation method of chicken Marek's virus antibody according to claim 1 and 2, it is characterized in that: the process of the standby yolk supernatant liquor of described employing sour water legal system is: with yolk, sodium-acetate one acetate buffer solution in 1:(6～14) ratio mix, filtered through gauze is to remove vitelline membrane, and be statically placed in 6～24h under the room temperature, make the yolk supernatant liquor, the pH value of described sodium-acetate-acetate buffer solution is 4.5～5.5, concentration is 0.05～0.12mol/L.
4. the preparation method of chicken Marek's virus antibody according to claim 1 and 2, it is characterized in that: it is the ultra-filtration membrane of 10～150KD that molecular weight cut-off is adopted in the described ultrafiltration that the yolk supernatant liquor is carried out, operation pressure reduction is 0.01～0.2MPa, and the concentrated solution volume that obtains is 1/5～1/2 of yolk volume.
5. the preparation method of chicken Marek's virus antibody according to claim 1 and 2, it is characterized in that: described employing chloroform to the process that concentrated solution carries out the degreasing purification process is: after adding final concentration and be 1～5% chloroform in the yolk supernatant liquor being carried out the concentrated solution that ultrafiltration obtains, concussion 3～5min, under 4 ℃ temperature condition, leave standstill 30min～72h, use again the centrifugal 10～60min of centrifugal force of 8000～10000 * g, collect supernatant liquor, and to regulate its pH value be 6.0～7.4, namely obtains the purpose antibody of clear.
6. according to the preparation method of the described chicken Marek's virus antibody of claim l, it is characterized in that: herpes turkey virus Fc-126 strain of the CVI988 strain that described Marek's disease virus vaccine strain is the serum I type or Serum III type; The serum I type MDV gentle strain of described pathogenic Mareks disease virus for can effectively secreting Mareks disease virus purpose antigen.
7. the preparation method of chicken Marek's virus antibody according to claim 1 and 2, it is characterized in that: the preparation method of described MDV antigen adjuvant vaccine is: the MDV antigen that will extract purifying carries out emulsification preparation and check with oil phase after being made into according to a conventional method water, and MDV antigen is MDV Staphylococal Protein A or MDV B antigen.
8. according to claim 1 to the chicken Marek's virus antibody of the preparation method of 7 arbitrary described chicken Marek's virus antibodies preparation.
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| CN103235129B (en) * | 2013-04-17 | 2015-10-28 | 河南省农业科学院 | Marek's disease virus and J subgroup avian leucosis virus fast joint inspection test strips |
| CN114656552B (en) * | 2021-12-17 | 2024-03-26 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | anti-Marek's virus monoclonal antibody, hybridoma cell strain and detection kit application |
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| CN101575374A (en) | 2009-11-11 |
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