CN104162161B - A kind of anti-duck viral hepatitis compositions, lyophilized powder, preparation method and application - Google Patents
A kind of anti-duck viral hepatitis compositions, lyophilized powder, preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of anti-duck viral hepatitis compositions, lyophilized powder, preparation method and application, belong to field of feed additive technology for animals.Said composition is made up of the component of following weight portion: anti-duck virus hepatitis yolk antibody 92～97 parts, anti-duck viral hepatitis transfer factor 1～3 parts.This lyophilized powder is made up of the component of following weight portion: anti-duck virus hepatitis yolk antibody 95～97 parts, anti-duck viral hepatitis transfer factor 1～2 parts, inactivator 0.2～0.3 part, preservative 0.01～0.02 part, heat-resisting lyophilized protecting agent 2.05～3.6 parts；There is reduction preservation condition, extend the holding time, the advantages such as antibody titer is high, biological activity is good, effect that anti-duck viral hepatitis transfer factor and anti-duck virus hepatitis yolk antibody are sick with the use of having preventing and treating duck viral hepatitis, immunity and the survival rate of duck, easy to use and low cost can be improved.
Description
Technical field
Present invention relates particularly to a kind of anti-duck viral hepatitis compositions, and anti-duck viral hepatitis lyophilized powder, simultaneously
Further relate to the preparation method and application of this lyophilized powder, belong to field of feed additive technology for animals.
Background technology
Duck occupies larger share in China's aquaculture, every year the amount of delivering for sale nearly 2,000,000,000, and meat yield reaches nearly 3,000,000
Ton, annual return reaches more than 30 hundred million.But, duck disease is wreaked havoc and is brought massive losses for China's aquaculture, wherein, and duck viral liver
Inflammation, is to endanger one of the most serious epidemic disease of duckling.
It is scorching that duck viral hepatitis is called for short duck liver, the duckling within main harm 20 age in days, and duckling age in days is the least, and mortality rate is more
Height, high mortality are up to 90～95%, and all of antibiotics is invalid to primary disease.Exist as far back as duck viral hepatitis in 1958
China occurs, it is the most popular in China that phase early 1980s plays primary disease, and since 1997, duck viral hepatitis is on some ground
District occurs more serious popular, and its epidemic situation fully can not be controlled by standard duck viral hepatitis I type attenuated vaccine, suspects there is I
Type virulent duck enteritis virus variant occurs.
At present, people carry out immunity by vaccine antagonism duck viral hepatitis disease, and early stage results is preferable, however it is necessary that many
Secondary inoculation, troublesome poeration, relatively costly, easily make poultry produce stress, and vaccine requires the most severe for storage conditions
Carving, need cold preservation at low temperatures, room temperature condition lower a period of time then lost efficacy.Therefore, duck viral can be prevented and treated in the urgent need to one
The product of hepatitis.
Summary of the invention
It is an object of the invention to provide a kind of anti-duck viral hepatitis compositions.
Meanwhile, the present invention also provides for a kind of anti-duck viral hepatitis lyophilized powder.
Furthermore, the present invention also provides for the preparation method of a kind of anti-duck viral hepatitis lyophilized powder.
Finally, the present invention also provides for the application in preparing feedstuff of a kind of anti-duck viral hepatitis lyophilized powder.
In order to realize object above, the technical solution adopted in the present invention is:
A kind of anti-duck viral hepatitis compositions, is made up of the component of following weight portion: anti-duck viral hepatitis yolk resists
Body 92～97 parts, anti-duck viral hepatitis transfer factor 1～3 parts.
A kind of anti-duck viral hepatitis lyophilized powder, is made up of the component of following weight portion: anti-duck viral hepatitis yolk resists
Body 95～97 parts, anti-duck viral hepatitis transfer factor 1～2 parts, inactivator 0.2～0.3 part, preservative 0.01～0.02 part,
Heat-resisting lyophilized protecting agent 2.05～3.6 parts.
Described inactivator is formaldehyde.
Described preservative is thimerosal.
Preferably, described heat-resisting lyophilized protecting agent is made up of the component of following weight portion: selenium 0.05～0.1 part, and dimension is raw
Element C0.5～1 part, polyvinylpyrrolidone 1～2 parts, glycine 0.5 part.
The preparation method of a kind of anti-duck viral hepatitis lyophilized powder, specifically comprises the following steps that
(1) isolated strain prepares vaccine: prepare vaccine after taking virulent duck enteritis virus strain propagation, inactivation standby；
(2) immune health chicken group prepares high-immunity egg: takes vaccine virus immunization healthy chicken flock prepared by step (1), works as antibody
When titer reaches 1:256, dry standby after collecting high-immunity egg cleaning, sterilization；
(3) acidification: take high-immunity egg separation yolk prepared by step (2), add isopyknic sterilized water, be sufficiently stirred for
Turning white to obtain yolk solution to color, add the pre-cooling acidifying water that pH value is 4.9～5.2 in yolk solution, addition is yolk
8 times of liquor capacity, staticly settle under room temperature after mixing；
(4) take the supernatant after step (3) stands, by volume add NaCl and NaSO of 0.8%4Mixture, anti-to improve
The degree of dissociation of body, the supernatant being centrifuged containing anti-duck virus hepatitis yolk antibody；
(5) taking the supernatant in step (4), in supernatant, the amount of anti-duck virus hepatitis yolk antibody is 95～97 parts,
The anti-duck viral hepatitis transfer factor of 1～2 part is added again, successively with 0.45 μm and the filter of 0.22 μm after mixing in supernatant
Membrane filtration；
(6) filtrate of step (5) is concentrated to give concentrated solution, be sequentially added in concentrated solution 0.2～0.3 part inactivator,
The preservative of 0.01～0.02 part and the heat-resisting lyophilized protecting agent of 2.05～3.6 parts, i.e. obtain anti-through pre-freeze, frozen dried after mixing
Duck viral hepatitis lyophilized powder.
Described step (2) uses inactivated vaccine normal amount for the first time, is 103.2ELD50/ 0.1ml duck viral hepatitis is sick
Poison inactivated vaccine 1.5mL, is spaced immunity in 7 days 1 time, and follow-up each immunity increases by 1 times amount, starts detection anti-after second time immunity
Body titer.
NaCl and NaSO in described step (4)4Mass ratio be 4:1.
In described step (5), anti-duck viral hepatitis transfer factor can be commercial goods, it would however also be possible to employ following methods
Prepare:
(1) pig spleen crossed with the normal saline cleaning duck viral hepatitis antigen immune after sterilizing, go its surface muscle,
The tissues such as fat, then use normal saline flushing；
(2) pig spleen after step (1) being processed crushes, and adds the PBS of 2 times of pre-coolings, will with tissue homogenate instrument
Its homogeneous homogenate；
(3) the homogenate Ultrasonic Cell Disruptor in step (2) is processed 10min and must crush liquid；
(4) the broken liquid of step (3) being adjusted to pH value is 5.3～5.7, and 6000rpm is centrifuged 30min, collects supernatant；
(5) by the supernatant micro-filtrate membrane filtration of step (4), the daltonian material of molecular cut off 7000, collect supernatant
Liquid；
(6) by the degerming membrane filtration of supernatant of step (5), anti-duck viral hepatitis transfer factor stock solution is obtained, 4 DEG C
Preserve or lyophilizing is standby.
Stock solution can be converted into transfer factor by the anti-duck viral hepatitis transfer factor added in lyophilized powder of the present invention
Content adds, it is also possible to add the transfer factor powder of lyophilised process.
The application in preparing feedstuff of a kind of anti-duck viral hepatitis lyophilized powder, step is as follows: take anti-duck viral hepatitis
Lyophilized powder, is 0.2～0.6% to add in feedstuff according to percentage by weight, mixes and get final product.
Beneficial effects of the present invention:
Feed additive of the present invention has reduction preservation condition with anti-duck viral hepatitis lyophilized powder, extends the holding time,
The advantages such as antibody titer is high, biological activity is good, add it by a certain percentage in feed formula, can prevent and treat duck sick
Virus hepatitis, improves immunity and the survival rate of duck, easy to use, can reduce cost and labour force.This lyophilized powder is by two kinds of tools
The biological substance having different bioactive anti-duck viral hepatitis merges, and can improve the energy of preventing and treating duck viral hepatitis
Power.
Wherein, antibody is to have relatively strong bioactive substance targetedly, is belonging to the bioactive substance passively acted on,
Can not effectively transfer the function the most actively of body, can only play a role corresponding virus, transfer factor is a kind of special
Regulation immune function active substance, the effective immune system function of body can be regulated, thus be actively engaged in immunity, thorn
Swash the immunity of the body fluid interareas such as lymphocyte.Both will be greatly improved attack and defense effect at combination, and that improves body prevents and treats function.
In the present invention anti-duck virus hepatitis yolk antibody can effective preventing duck virus hepatitis, animal is not had that poison is secondary to be made
With, environmental protection, noresidue, height is exempted from the anti-duck virus hepatitis yolk antibody for preparing and adds in feedstuff as addO-on therapy, can be
Go up preventing duck virus hepatitis largely, strengthen addition and can also treat duck viral hepatitis；Anti-duck viral hepatitis shifts
The factor and anti-duck virus hepatitis yolk antibody are with the use of the prevention effect that can improve folk prescription.
The present invention uses healthy chicken flock as immunization, can reduce the probability of strain reversion, can quickly carry simultaneously
Duck viral hepatitis prevention effect, to 1:256, is highlighted by the yolk antibody made after chicken reinforced immunological, sees by high antibody titer
Imitate fast, evident in efficacy and with strong points.
In the present invention anti-duck viral hepatitis lyophilized powder, heat-resisting lyophilized protecting agent uses selenium, Vc, polyvinylpyrrolidone
(PVP) and four kinds of components of glycine, wherein, selenium is a kind of trace element, and tool antimutagenic effect, antioxidation, promotion are carcinogenic
The internal deactivation of thing, cell proliferation effect, enhancing human body immunity power effect, regulate and improve the work of body's immunity
With, make body specific immunity and nonspecific immunity, humoral immunization and cellular immune function be in relative balance state, have
Anti-aging effects, can slow down the effective active of bioactive substance；The effect of Vc has a variety of, has non-oxidizability and keeps away
Exempt from a series of change after Exposure to Sunlight, it is also possible to the regulation variations in temperature adaptation ability to body, irony can also be promoted simultaneously
With calcareous Absorption；PVP is the adhesive of medicine, solubilizing agent, disintegrating agent, prolongation of effect agent, utilizes PVP molecule and drug molecule
Between hydrogen bond association can play and contrary prolongations of effect solubilized, quick-acting, slow releasing function, control PVP molecule and drug molecule
Between associating intensity, after can making association, drug molecule discharges in human body lentamente, delays its dissolution velocity, plays and prolong
The effect of long drug effect ground, has obvious biological protection function simultaneously；Glycine has anticorrosion, cushioning effect, it is also possible to play steady
Determine the effect of agent.
Detailed description of the invention
The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.
Embodiment 1
The anti-duck viral hepatitis compositions of the present embodiment, is made up of the component of following weight portion: anti-duck viral hepatitis
Yolk antibody 97 parts, anti-duck viral hepatitis transfer factor 1 part.
The anti-duck viral hepatitis lyophilized powder of the present embodiment, is made up of the component of following weight portion: anti-duck viral hepatitis
Yolk antibody 97 parts, anti-duck viral hepatitis transfer factor 1 part, 0.2 part of formaldehyde, thimerosal 0.01 part, 0.05 part of selenium, vitamin
C0.5 part, polyvinylpyrrolidone 1 part, glycine 0.5 part.
In the present embodiment, the preparation method of anti-duck viral hepatitis lyophilized powder, specifically comprises the following steps that
(1) strain prepares vaccine: prepare emulsifiable concentrate vaccine after taking national standard virulent duck enteritis virus strain propagation, inactivation
Standby；
(2) immune health chicken group prepares high-immunity egg: take emulsifiable concentrate vaccine virus immunization healthy chicken flock prepared by step (1),
Use inactivated vaccine normal amount for the first time, be 103.2ELD50/ 0.1ml virulent duck enteritis virus inactivated vaccine 1.5mL, is spaced 7
It immunity 1 time, follow-up each immunity increases by 1 times amount, and immunity three times, start to detect antibody titer after second time immunity altogether, when
When antibody titer reaches 1:256, starting to collect high-immunity egg, high-immunity egg dries standby after cleaning, sterilization；
(3) acidification: take high-immunity egg separation yolk prepared by step (2), add isopyknic sterilized water, be sufficiently stirred for
Turning white to obtain yolk solution to color, add the pre-cooling acidifying water that pH value is 4.9 in yolk solution, addition is yolk solution body
Long-pending 8 times, mixing is placed in storage tank and staticly settles at room temperature；
(4) take the supernatant after step (3) stands, by volume add NaCl and NaSO of 0.8%4(NaCl and NaSO4Matter
Amount ratio is 4:1) mixture, to improve the degree of dissociation of antibody, more centrifugal 20min must be containing anti-duck virus under 15000rpm/min
The supernatant of property hepatitis yolk antibody；
(5) taking the supernatant in step (4), in supernatant, the amount of anti-duck virus hepatitis yolk antibody is 97 parts, then
Adding 1 part of anti-duck viral hepatitis transfer factor in supernatant, with 0.45 μm membrane filtration after mixing, to dam, molecular weight is more than
The macromolecular substances of 200KD, takes filtrate with 0.22 μm filter membrane secondary filter；
(6) through ultrafiltration apparatus, the filtrate of step (5) being concentrated into yolk antibody concentration is 2.5mL/500 plumage part, is concentrating
Liquid is sequentially added into 0.2 part of formaldehyde, 0.01 part of thimerosal, 0.05 part of selenium, 0.5 part of vitamin C, 1 part of polyvinylpyrrolidone,
0.5 part of glycine, mixing is fully freezing at being placed on-80 DEG C, and is placed in freeze dryer lyophilizing and get final product.
In the present embodiment, the preparation method of anti-duck viral hepatitis transfer factor is:
(1) pig spleen crossed with the normal saline cleaning duck viral hepatitis antigen immune after sterilizing, go its surface muscle,
The tissues such as fat, then use normal saline flushing；
(2) pig spleen after step (1) being processed crushes, and adds the PBS of 2 times of pre-coolings, will with tissue homogenate instrument
Its homogeneous homogenate；
(3) the homogenate Ultrasonic Cell Disruptor in step (2) is processed 10min and must crush liquid；
(4) the broken liquid of step (3) being adjusted to pH value is 5.5, and 6000rpm is centrifuged 30min, collects supernatant；
(5) by the supernatant micro-filtrate membrane filtration of step (4), the daltonian material of molecular cut off 7000, collect supernatant；
(6) by the degerming membrane filtration of supernatant of step (5), obtain anti-duck viral hepatitis transfer factor stock solution, freeze
Dry standby.
The application in preparing feedstuff of the anti-duck viral hepatitis lyophilized powder of the present embodiment, step is as follows: take anti-duck virus
Property hepatitis lyophilized powder, is in 0.2% addition feedstuff according to percentage by weight, mixes and get final product.
Embodiment 2
The anti-duck viral hepatitis compositions of the present embodiment, is made up of the component of following weight portion: anti-duck viral hepatitis
Yolk antibody 95 parts, anti-duck viral hepatitis transfer factor 1.5 parts.
The anti-duck viral hepatitis lyophilized powder of the present embodiment, is made up of the component of following weight portion: anti-duck viral hepatitis
Yolk antibody 95 parts, anti-duck viral hepatitis transfer factor 1.5 parts, 0.3 part of formaldehyde, thimerosal 0.02 part, 0.1 part of selenium, dimension is raw
Element C1 part, polyvinylpyrrolidone 2 parts, glycine 0.5 part.
In the present embodiment, the preparation method of anti-duck viral hepatitis lyophilized powder, specifically comprises the following steps that
(1) strain prepares vaccine: prepare emulsifiable concentrate vaccine after taking national standard virulent duck enteritis virus strain propagation, inactivation
Standby；
(2) immune health chicken group prepares high-immunity egg: take emulsifiable concentrate vaccine virus immunization healthy chicken flock prepared by step (1),
Use inactivated vaccine normal amount for the first time, be 103.2ELD50/ 0.1ml virulent duck enteritis virus inactivated vaccine 1.5mL, is spaced 7
It immunity 1 time, follow-up each immunity increases by 1 times amount, and immunity three times, start to detect antibody titer after second time immunity altogether, when
When antibody titer reaches 1:256, starting to collect high-immunity egg, high-immunity egg dries standby after cleaning, sterilization；
(3) acidification: take high-immunity egg separation yolk prepared by step (2), add isopyknic sterilized water, be sufficiently stirred for
Turning white to obtain yolk solution to color, add the pre-cooling acidifying water that pH value is 5.2 in yolk solution, addition is yolk solution body
Long-pending 8 times, mixing is placed in storage tank and staticly settles at room temperature；
(4) take the supernatant after step (3) stands, by volume add NaCl and NaSO of 0.8%4(NaCl and NaSO4Matter
Amount ratio is 4:1) mixture, to improve the degree of dissociation of antibody, more centrifugal 20min must be containing anti-duck virus under 15000rpm/min
The supernatant of property hepatitis yolk antibody；
(5) taking the supernatant in step (4), in supernatant, the amount of anti-duck virus hepatitis yolk antibody is 95 parts, then
Supernatant adds 1.5 parts of anti-duck viral hepatitis transfer factors (preparation method is with embodiment 1), with 0.45 μm filter membrane after mixing
Filter, with the molecular weight that dams more than the macromolecular substances of 200KD, take filtrate with 0.22 μm filter membrane secondary filter；
(6) through ultrafiltration apparatus, the filtrate of step (5) being concentrated into yolk antibody concentration is 2.5mL/500 plumage part, is concentrating
Liquid is sequentially added into 0.3 part of formaldehyde, 0.02 part of thimerosal, 0.1 part of selenium, 1 part of vitamin C, 2 parts of polyvinylpyrrolidones, 0.5 part
Glycine, mixing is fully freezing at being placed on-80 DEG C, and is placed in freeze dryer lyophilizing and get final product.
The application in preparing feedstuff of the anti-duck viral hepatitis lyophilized powder of the present embodiment, step is as follows: take anti-duck virus
Property hepatitis lyophilized powder, is in 0.4% addition feedstuff according to percentage by weight, mixes and get final product.
Embodiment 3
The anti-duck viral hepatitis compositions of the present embodiment, is made up of the component of following weight portion: anti-duck viral hepatitis
Yolk antibody 96 parts, anti-duck viral hepatitis transfer factor 2 parts.
The anti-duck viral hepatitis lyophilized powder of the present embodiment, is made up of the component of following weight portion: anti-duck viral hepatitis
Yolk antibody 96 parts, anti-duck viral hepatitis transfer factor 2 parts, 0.25 part of formaldehyde, thimerosal 0.015 part, 0.075 part of selenium, dimension
Raw element C0.75 part, polyvinylpyrrolidone 1.5 parts, glycine 0.5 part.
In the present embodiment, the preparation method of anti-duck viral hepatitis lyophilized powder, specifically comprises the following steps that
(1) strain prepares vaccine: prepare emulsifiable concentrate vaccine after taking national standard virulent duck enteritis virus strain propagation, inactivation
Standby；
(2) immune health chicken group prepares high-immunity egg: take emulsifiable concentrate vaccine virus immunization healthy chicken flock prepared by step (1),
Use inactivated vaccine normal amount for the first time, be 103.2ELD50/ 0.1ml virulent duck enteritis virus inactivated vaccine 1.5mL, is spaced 7
It immunity 1 time, follow-up each immunity increases by 1 times amount, and immunity three times, start to detect antibody titer after second time immunity altogether, when
When antibody titer reaches 1:256, starting to collect high-immunity egg, high-immunity egg dries standby after cleaning, sterilization；
(3) acidification: take high-immunity egg separation yolk prepared by step (2), add isopyknic sterilized water, be sufficiently stirred for
Turning white to obtain yolk solution to color, add the pre-cooling acidifying water that pH value is 5.0 in yolk solution, addition is yolk solution body
Long-pending 8 times, mixing is placed in storage tank and staticly settles at room temperature；
(4) take the supernatant after step (3) stands, by volume add NaCl and NaSO of 0.8%4(NaCl and NaSO4Matter
Amount ratio is 4:1) mixture, to improve the degree of dissociation of antibody, more centrifugal 20min must be containing anti-duck virus under 15000rpm/min
The supernatant of property hepatitis yolk antibody；
(5) taking the supernatant in step (4), in supernatant, the amount of anti-duck virus hepatitis yolk antibody is 96 parts, then
Supernatant adds 2 parts of anti-duck viral hepatitis transfer factors (preparation method is with embodiment 1), by 0.45 μm filter membrane mistake after mixing
Filter, with the molecular weight that dams more than the macromolecular substances of 200KD, takes filtrate with 0.22 μm filter membrane secondary filter；
(6) through ultrafiltration apparatus, the filtrate of step (5) being concentrated into yolk antibody concentration is 2.5mL/500 plumage part, is concentrating
Liquid is sequentially added into 0.25 part of formaldehyde, 0.015 part of thimerosal, 0.075 part of selenium, 0.75 part of vitamin C, 1.5 parts of polyvinyl pyrrole
Alkanone, 0.5 part of glycine, mixing is fully freezing at being placed on-80 DEG C, and is placed in freeze dryer lyophilizing and get final product.
The application in preparing feedstuff of the anti-duck viral hepatitis lyophilized powder of the present embodiment, step is as follows: take anti-duck virus
Property hepatitis lyophilized powder, is in 0.6% addition feedstuff according to percentage by weight, mixes and get final product.
Comparative example 1
The feed additive of this comparative example is with anti-duck virus hepatitis yolk antibody lyophilized powder, by the component of following weight portion
Composition: anti-duck virus hepatitis yolk antibody 98 parts, 0.25 part of formaldehyde, thimerosal 0.015 part, 0.075 part of selenium, vitamin
C0.75 part, polyvinylpyrrolidone 1.5 parts, glycine 0.5 part.The preparation method and application of lyophilized powder are with embodiment 3.
Comparative example 2
The feed additive of this comparative example is with anti-duck viral hepatitis transfer factor lyophilized powder, by the component of following weight portion
Composition: anti-duck viral hepatitis transfer factor 98 parts, 0.25 part of formaldehyde, thimerosal 0.015 part, 0.075 part of selenium, vitamin
C0.75 part, polyvinylpyrrolidone 1.5 parts, glycine 0.5 part.
Test example 1
Experiment of preventive effects: use 7 age in days duckling 700, wherein 100 is only used as comparison isolation and supports, use common raising
Material；100 are only used as blank group, use normal diet；Another 500 test isolated rearings, are randomly divided into 5 groups, often group 100,
Using embodiment 1～3 and the feedstuff of comparative example 1～2 preparation, feed ingredient is organized consistent with matched group and blank.Feeding manner is
Free choice feeding, freely drinks water, and after 24h, takes DHV virus liquid after the duckling intramuscular injection inoculation dilution in test group and matched group, record
120h death condition, result of the test refers to table 1 below.
Therapeutic effect is tested: use 7 age in days duckling 600, the DHV virus liquid after 600 eye dripping inoculation dilutions, raising side
Formula is free choice feeding and freely drinks water, and after infecting 12h, wherein 100 is only used as positive controls, uses normal diet；Another 500
For test group, it is randomly divided into 5 groups, often group 100, uses embodiment 1～3 and the feedstuff of comparative example 1～2 preparation, feed ingredient
Consistent with positive controls, that record 120h respectively organizes continuously death condition, result of the test refers to table 1 below.
The result of table 1 each process group preventing and treating duck viral hepatitis
| Experiment of preventive effects | Therapeutic effect is tested |
| Group | Death toll/only | Protective rate/% | Death toll/only | Survival rate/% |
| Embodiment 1 | 14 | 86% | 14 | 86% |
| Embodiment 2 | 8 | 92% | 7 | 93% |
| Embodiment 3 | 5 | 95% | 4 | 96% |
| Comparative example 1 | 20 | 80% | 35 | 65% |
| Comparative example 2 | 25 | 75% | 30 | 70% |
| Counteracting toxic substances matched group | 85 | 15% | 86 | 14% |
| Blank group | 0 | 100% | / | / |
Conclusion: as shown in Table 1, the feedstuff of embodiment 1～3 preparation has good prophylactic-therapeutic effect to duck viral hepatitis,
Protective rate and survival rate are all more than 86%, and the feedstuff prevention effect of comparative example 1～2 preparation is not as embodiment.
Test example 2
By embodiment 1～3 and the lyophilized powder prepared of comparative example 1 be saved in 37 DEG C of incubators, preserve 24 months, respectively at
After placement 1 week, 2 weeks, January, February, April, August, December, 16 months, 20 months, 24 months, use agar diffusion method (AGP) to measure every
The titer of anti-duck virus hepatitis yolk antibody in individual time point sample, testing result refers to table 2 below.
The lyophilized powder change at different time sections antibody titer placed by 237 DEG C of table
| Group/titer | 1 week | 2 weeks | January | February | April | August | December | 16 months | 20 months | 24 months |
| Embodiment 1 | 1:32 | 1:32 | 1:32 | 1:32 | 1:32 | 1:16 | 1:16 | 1:16 | 1:16 | 1:16 |
| Embodiment 2 | 1:32 | 1:32 | 1:32 | 1:32 | 1:32 | 1:32 | 1:16 | 1:16 | 1:16 | 1:16 |
| Embodiment 3 | 1:32 | 1:32 | 1:32 | 1:32 | 1:32 | 1:32 | 1:16 | 1:16 | 1:16 | 1:16 |
| Comparative example 1 | 1:32 | 1:32 | 1:32 | 1:32 | 1:32 | 1:16 | 1:16 | 1:16 | 1:16 | 1:16 |
Conclusion, as shown in Table 2, the storage life of the biological effectiveness of the lyophilized powder of the embodiment of the present invention 1～3 preparation at least two years.
Quality standard
Several as described below of the quality standard of the lyophilized powder of embodiment 1～3 preparation:
Character: faint yellow, in fine and close agglomerate.
Mycoplasma is checked: carry out by " Chinese veterinary pharmacopoeia ", grows without mycoplasma.
Exogenous virus is checked: carries out by " Chinese veterinary pharmacopoeia ", meets regulation.
Safety verification: with 7 Japanese instar chickling 5, intramuscular injection 5 plumage part, observe 2 weeks, the most strong alive, injection site does not has pathological changes.
Residual moisture measures: carry out by " Chinese veterinary pharmacopoeia ", meets veterinary biologics general rule regulation.
Shelf-life: according to room temperature bioactivity experimental result, 2 years can be deposited.
Claims (6)
1. an anti-duck viral hepatitis lyophilized powder, it is characterised in that: include the component of following weight portion: anti-duck viral hepatitis
Yolk antibody 95～97 parts, anti-duck viral hepatitis transfer factor 1～2 parts, inactivator 0.2～0.3 part, preservative 0.01～
0.02 part, heat-resisting lyophilized protecting agent 2.05～3.6 parts；Described inactivator is formaldehyde, and preservative is thimerosal, and heat resistant freeze drying is protected
Protect agent to be made up of the component of following weight portion: selenium 0.05～0.1 part, vitamin C 0.5～1 part, polyvinylpyrrolidone 1～2
Part, glycine 0.5 part.
2. the preparation method of an anti-duck viral hepatitis lyophilized powder as claimed in claim 1, it is characterised in that: concrete steps
As follows:
(1) isolated strain prepares vaccine: prepare vaccine after taking virulent duck enteritis virus strain propagation, inactivation standby；
(2) immune health chicken group prepares high-immunity egg: takes vaccine virus immunization healthy chicken flock prepared by step (1), works as antibody titer
When reaching 1:256, dry standby after collecting high-immunity egg cleaning, sterilization；
(3) acidification: take high-immunity egg separation yolk prepared by step (2), add isopyknic sterilized water, be stirred well to face
Color turns white to obtain yolk solution, adds the acidifying water that pH value is 4.9～5.2 in yolk solution, and addition is yolk solution volume
8 times, staticly settle after mixing；
(4) take the supernatant after step (3) stands, by volume add NaCl and Na of 0.8%2SO4Mixture, so centrifugal that to contain
The supernatant of anti-duck virus hepatitis yolk antibody；
(5) taking the supernatant in step (4), in supernatant, the amount of anti-duck virus hepatitis yolk antibody is 95～97 parts, then
Supernatant adds the anti-duck viral hepatitis transfer factor of 1～2 part, successively by the filter membrane mistake of 0.45mm and 0.22mm after mixing
Filter；
(6) filtrate of step (5) is concentrated to give concentrated solution, be sequentially added in concentrated solution 0.2～0.3 part inactivator, 0.01
～the preservative of 0.02 part and the heat-resisting lyophilized protecting agent of 2.05～3.6 parts, i.e. obtain anti-duck through pre-freeze, frozen dried after mixing sick
Virus hepatitis lyophilized powder.
The preparation method of anti-duck viral hepatitis lyophilized powder the most according to claim 2, it is characterised in that: described step
(2) use inactivated vaccine normal amount in for the first time, be 103.2ELD50/ 0.1 ml virulent duck enteritis virus inactivated vaccine 1.5mL,
Being spaced immunity in 7 days 1 time, follow-up each immunity increases by 1 times amount, starts to detect antibody titer after second time immunity.
The preparation method of anti-duck viral hepatitis lyophilized powder the most according to claim 2, it is characterised in that: described step
(4) NaCl and Na in2SO4Mass ratio be 4:1.
The preparation method of anti-duck viral hepatitis lyophilized powder the most according to claim 2, it is characterised in that: described step
(5) in, the preparation method of anti-duck viral hepatitis transfer factor is:
(1) pig spleen crossed with the normal saline cleaning duck viral hepatitis antigen immune after sterilizing, removes its surface muscle, fat
In tissue, then use normal saline flushing；
(2) pig spleen after step (1) being processed crushes, and adds the PBS of 2 times of pre-coolings, with tissue homogenate instrument, it is even
Matter obtains homogenate；
(3) the homogenate Ultrasonic Cell Disruptor in step (2) is processed 10min and must crush liquid；
(4) the broken liquid of step (3) being adjusted to pH value is 5.3～5.7, and 6000rpm is centrifuged 30min, collects supernatant；
(5) by the supernatant micro-filtrate membrane filtration of step (4), the daltonian material of molecular cut off 7000, collect supernatant；
(6) by the degerming membrane filtration of supernatant of step (5), anti-duck viral hepatitis transfer factor stock solution, 4 DEG C of preservations are obtained
Or lyophilizing is standby.
6. an anti-duck viral hepatitis lyophilized powder as claimed in claim 1 application in preparing feedstuff, it is characterised in that:
Step is as follows: take anti-duck viral hepatitis lyophilized powder, is 0.2～0.6% to add in feedstuff according to percentage by weight, mixes and get final product.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101704890A (en) * | 2009-11-13 | 2010-05-12 | 烟台绿叶动物保健品有限公司 | Preparation method of duck hepatitis antibody |
| CN101953849A (en) * | 2010-10-19 | 2011-01-26 | 郑州后羿制药有限公司 | Method for preparing anti-duck viral hepatitis transfer factor |
| CN102241771A (en) * | 2011-05-19 | 2011-11-16 | 湖南临武舜华鸭业发展有限责任公司 | Duck egg yolk antibody for preventing and treating virus hepatitis of duckling, and preparation method thereof |
| CN102716484A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Duck virus hepatitis yolk antibody freeze-dried powder and preparation method thereof |
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2013
- 2013-07-24 CN CN201310315435.0A patent/CN104162161B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101704890A (en) * | 2009-11-13 | 2010-05-12 | 烟台绿叶动物保健品有限公司 | Preparation method of duck hepatitis antibody |
| CN101953849A (en) * | 2010-10-19 | 2011-01-26 | 郑州后羿制药有限公司 | Method for preparing anti-duck viral hepatitis transfer factor |
| CN102241771A (en) * | 2011-05-19 | 2011-11-16 | 湖南临武舜华鸭业发展有限责任公司 | Duck egg yolk antibody for preventing and treating virus hepatitis of duckling, and preparation method thereof |
| CN102716484A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Duck virus hepatitis yolk antibody freeze-dried powder and preparation method thereof |
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