CN105597093A - Preparation method of compound inactivated vaccine for newcastle disease - Google Patents

Preparation method of compound inactivated vaccine for newcastle disease Download PDF

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Publication number
CN105597093A
CN105597093A CN201511006408.0A CN201511006408A CN105597093A CN 105597093 A CN105597093 A CN 105597093A CN 201511006408 A CN201511006408 A CN 201511006408A CN 105597093 A CN105597093 A CN 105597093A
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parts
preparation
newcastle disease
cell
inactivated vaccine
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沈建军
张秀文
李阳
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ZHEJIANG MIBOLERONE BIOLOGICAL TECHNOLOGY Co Ltd
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ZHEJIANG MIBOLERONE BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The invention discloses a preparation method of a compound inactivated vaccine for newcastle disease and belongs to the technical field of veterinary biological products. The preparation method comprises the following steps of firstly, passage and culture of master cells; secondly, reproduction of a cell adapting virus seed; thirdly, large-scale culture of cells for preparing the vaccine; fourthly, preparation of venom for preparing the vaccine; fifthly, preparation of the compound inactivated vaccine for the newcastle disease. According to the preparation method disclosed by the invention, a new vaccine adjuvant is adopted; the prepared vaccine has double characteristics of water in oil and oil in water and has the advantages of sustained release action of the vaccine, and combined action of a water-soluble immune enhancer, an egg yolk antibody and the like; by adopting the effective immune enhancer, the immunity of an organism is greatly improved, the immunity of body fluid is improved, the organism is stimulated to generate a high-efficiency neutralizing antibody, and further the capability of the organism to prevent diseases is improved.

Description

The preparation method of the compound inactivated vaccine of a kind of newcastle disease
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to the preparation method of the compound inactivated vaccine of a kind of newcastle disease.
Background technology
Ewcastle disease (Newcastledisease, ND) be by NDV (Newcastlediseasevirus, NDV) a kind of potent virus sexually transmitted disease causing, it is one of fowl infection the most serious in current global range, in all chicken diseases of Ye Shi China, endanger maximum one, classified as category-A infectious disease by International Office of Epizootics (OIE).
Water-in-oil-in-water emulsion adjuvant has the feature of aluminium glue adjuvant, oil-in-water adjuvant and Water-In-Oil adjuvant, compared with water-in-oil emulsion adjuvant, it is prepared the needed antigen concentration of vaccine and greatly reduces, there is in addition the characteristic of oil-in-water adjuvant because of it, make it can add water miscible immunopotentiator, herbal polysaccharide and analgestic (vitamin), can alleviating pain in the time of vaccine organism, reduce Animal stress, immunopotentiator, herbal polysaccharide also have raising immune effect, the functions such as prevent disease simultaneously.
Polysaccharide is one of active ingredient of Chinese herbs, and quantity research shows greatly, and polysaccharide and saccharide complex are not only in vivo as energy resource and constituent material, the more important thing is that it is present in all membrane structures, participates in the comings and goings of cell in biological phenomena. Polysaccharose substance is the important component part of all living organisms, has the ability of removing free radical, improving activities of antioxidant enzymes and inhibition lipid peroxidation.
Vitamin is requisite organic compound in organism metabolism. Body, just as a very complicated chemical plant, is constantly carrying out various biochemical reactions. Its reaction has substantial connection with the catalytic action of enzyme. Enzyme will produce activity, must have coenzyme to participate in. Known much vitamin is the coenzyme of enzyme or the ingredient of coenzyme. Therefore, vitamin is the important substance that maintains and regulate body eubolism.
Bursopoietin (bursin, BS) is a kind of active kyrine material of separating from the distinctive humoral immunity central lymphoid organs of the bird bursa of farbricius (Bursaoffabricius), and its structure is L-Lys-His-Gly-NH2. Research shows, BS can promote differentiation, the propagation of bird and mammal bone-marrow-derived lymphocyte precursor, can improve second messenger cAMP and cGMP level in B clone Daudi cell, accelerate the speed that the interior DNA of B cell is transcribed into mRNA, promote the generation of B intracellular protein, thereby B cell is produced and the reinforcement of secretory antibody ability.
The structure of Yolk antibody (IgY) molecule and mammalian immune Globulins are seemingly. Yolk antibody is as a kind of immunoglobulin (Ig) with BA, producing, processing, store and ingest and digestion process in, it is crucial keeping the activity of antibody. Multinomial experiment shows that chicken IgY has good stability, acidproof, heat-resisting. IgY preserves 6 months at room temperature, and its virus neutralizing cpaacity is without obvious change. Li Chunhui etc. (2002) have studied the impact of pepsin antagonism rabies viruses IgY activity. Result show IgY after pepsin enzymolysis 24h, still keep in and the effect of antigen, its enzymatic fragment also has antibody activity. Yolk antibody also has unrivaled superiority on producing. First, produce the required antigen amount of effective immune response little, especially in the mammalian proteins confrontation phylogenetics of high conservative, distant bird has stronger immunogenicity conventionally. Secondly, collect egg instant, without blood sampling, not damaged, meets modern animal protection rule. Europe alternative authentication center (ECVAM) suggestion replaces the source of mammal IgG as experiment and production antibody using IgY. The 3rd, each egg is approximately containing more than 100mg IgY, within one month, can reach 10-20 that 3g is equivalent to rabbit doubly; In yolk, there is no other Ig, be easy to purify. The 4th, owing to being subject to the protection of yolk, egg is stored in lower 6 months of 4 DEG C of conditions, and IgY loss of activity is very little. So can collect the IgY that purifies on a large scale when more a certain amount of until egg.
The present invention is based on above-mentioned technical background, propose a kind of preparation method of newcastle disease inactivated vaccine, this vaccine, by newcastle disease inactivation antigen, adds the appropriate immunopotentiator that contains and makes, and formulation is W/O/W type. The compound inactivated vaccine of preparing by the method, because it has oil-in-water, Water-In-Oil two specific characters, make the immunopotentiators such as water miscible important flavones, bursopoietin, Yolk antibody and analgestic be easy to dissolve, being beneficial to animal body absorbs, when immunity, can reduce Animal stress, reduce animal suffering, can improve animal welfare; Immunopotentiator can improve the cellular immunity of inactivated vaccine greatly simultaneously, produces higher neutralizing antibody, significantly improves protective rate.
Summary of the invention
The problem existing for prior art, the object of the invention is to design provides the preparation method's of the compound inactivated vaccine of a kind of newcastle disease technical scheme, adopt this method to prepare vaccine, be characterized in that prepared virus titer is high, prepared vaccine differences between batches are little, constant product quality is efficient, and side reaction is little, can improve vaccine quality comprehensively.
The preparation method of the described compound inactivated vaccine of a kind of newcastle disease, is characterized in that comprising following processing step:
(1) plant going down to posterity and cultivating of cell: chicken embryo continuous cell line, through pancreatin cell dispersion liquid had digestive transfer culture, continues to cultivate with cell growth medium, while forming good individual layer, goes down to posterity or virus inoculation for continuing;
(2) breeding of cell adapted seed culture of viruses: get cell adapted NDV seed culture of viruses, be inoculated in the chicken embryo passage cell that has grown up to individual layer in step (1), put 37～38 DEG C and continue to cultivate, cytopathy variability reaches 75% liquid of harvesting when above;
(3) large-scale culture of cell for seedling: step (1) is cultivated to the kind cell suspending liquid obtaining and be seeded in rolling bottle or cell factory, add cell growth medium to put 37～38 DEG C and cultivate;
(4) preparation of venom for seedling: get the above-mentioned clone blake bottle that has formed good individual layer in step (3), discard cell growth medium, the cell adapted malicious viral suspension of inoculation ewcastle disease, after absorption, add maintenance medium, putting 37～38 DEG C continues to cultivate, when reaching 75%, cytopathy gathers in the crops venom when above ,-15 DEG C of following preservations;
(5) the compound inactivated vaccine preparation of newcastle disease: the virus liquid obtaining in step (4) is carried out to steriling test, viral level mensuration, carry out after the assay was approved formalin-inactivated, deactivation after the assay was approved, in the Newcastle Disease Virus Antigen liquid being up to the standards in every 1000ml deactivation, add 1～2 portion of immunopotentiator to mix, then with Sang meter Te domestic animal W/O/W adjuvant by volume 3:2 carry out emulsification packing and make the compound inactivated vaccine of newcastle disease.
The preparation method of the described compound inactivated vaccine of a kind of newcastle disease, it is characterized in that in described step 4), every portion of immunopotentiator contains herbal polysaccharide 20～50g, vitamin combination 6～10g, bursopoietin 2～16mg and Yolk antibody 5～25g, be preferably herbal polysaccharide 30～40g, vitamin combination 7～9g, bursopoietin 8～12mg and Yolk antibody 10～20g.
The preparation method of the described compound inactivated vaccine of a kind of newcastle disease, is characterized in that described herbal polysaccharide makes by following steps:
A, press 10～30 parts of ginsengs, 10～30 parts, Poria cocos, 20～40 parts of plantain seeds, 10～20 parts of the Radixs Astragali, 10～30 parts, Radix Glycyrrhizae, 20～30 parts of pawpaws, 10～20 parts of gingkoes, 10～30 parts of reed rhizomes take each Chinese medicine material, chopping, spend the night by cold water soak after cleaning, then add the purified water of 15 times of raw material weights, water-bath to 90 DEG C, and remain on 90 DEG C, and boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect liquid, after room temperature is cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out to alcohol precipitation, precipitation is rough herbal polysaccharide;
D, with rough herbal polysaccharide in sterilizing purified water washing c, after washing, dissolves with 42 DEG C of sterilizing purified water of 20 times of amounts, after dissolving, add 4 DEG C of absorption of active carbon to spend the night in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, by supernatant in d after the degerming of 0.22um membrane filtration, carry out 10 times concentrated, make herbal polysaccharide concentrate;
F, herbal polysaccharide concentrate in e is carried out to vacuum freeze drying obtain herbal polysaccharide.
The preparation method of the described compound inactivated vaccine of a kind of newcastle disease, is characterized in that containing B615～35% of supporting one's family, folic acid 40～70% and vitamin C 10～30% in described vitamin combination.
The preparation method of the described compound inactivated vaccine of a kind of newcastle disease, it is characterized in that described bursopoietin makes by following steps: bursa of farbricius tissue is rejected to manadesma and adipose tissue, the cold PBS of pH7.2 sterilizing cleans, add the cold PBS of pH7.2 sterilizing in 1:1 ratio, in tissue refiner, carry out high-speed homogenization, in homogenate, add the trypsase that accounts for 2.5% weight, multigelation 3 times, the centrifugal 20min of 12000rpm, abandon precipitation, supernatant carries out ultrafiltration with the milipore filter of 1000da molecular cut off, under film, liquid is through the degerming of 0.22um membrane filtration, filtered solution is bursopoietin crude extract, crude extract obtains bursopoietin through vacuum freezedrying.
The preparation method of the described compound inactivated vaccine of a kind of newcastle disease, is characterized in that described Yolk antibody adopts newcastle disease efficient concentration inactivated vaccine Immune Laying Hens, separates yolk, and acidifying makes through vacuum freeze drying after extracting.
The preparation method of the described compound inactivated vaccine of a kind of newcastle disease, it is characterized in that in described step a by 12～26 parts of ginsengs, 12～26 parts, Poria cocos, 25～35 parts of plantain seeds, 12～18 parts of the Radixs Astragali, 15～25 parts, Radix Glycyrrhizae, 22～28 parts of pawpaws, 12～18 parts of gingkoes, 15～50 parts of reed rhizomes take each Chinese medicine material.
In the present invention, Sang meter Te domestic animal W/O/W adjuvant is by Zhuhai Guo Nian bio tech ltd production and sales.
The present invention has following beneficial effect:
1. the present invention has adopted new vaccine adjuvant, and prepared vaccine has Water-In-Oil, oil-in-water double grading, has had the coefficient advantages such as the slow releasing function of vaccine and water-soluble immunopotentiator, Yolk antibody concurrently.
2. the present invention has adopted effective immunopotentiator, has increased substantially the immunity of body, improves humoral immunity ability, stimulates body to produce efficient neutralizing antibody, has improved the prophylactic ability of body.
Detailed description of the invention
In order to make the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention. Should be understood that these embodiment are only not used in the scope of the present invention of placing restrictions on for the present invention is described, NM specific experiment method in the following example, experimental technique carries out routinely conventionally.
The preparation of embodiment 1 newcastle disease inactivation of viruses suspension
(1) plant going down to posterity and cultivating of cell: chicken embryo continuous cell line is through pancreatin cell dispersion liquid had digestive transfer culture, continue to cultivate with cell growth medium, while forming good individual layer, go down to posterity or virus inoculation for continuing, cell growth medium is the DMEM/F12 solution containing 10% hyclone;
(2) breeding of cell adapted seed culture of viruses: get cell adapted NDV seed culture of viruses, be inoculated in the chicken embryo passage cell that has grown up to individual layer in step (1), put 37～38 DEG C and continue to cultivate, cytopathy variability reaches 75% liquid of harvesting when above;
(3) large-scale culture of cell for seedling: step (1) is cultivated to the kind cell suspending liquid obtaining and be seeded in rolling bottle or cell factory, add cell growth medium to put 37～38 DEG C and cultivate;
(4) preparation of venom for seedling: get the above-mentioned clone blake bottle that has formed good individual layer in step (3), discard cell growth medium, the cell adapted malicious viral suspension of inoculation ewcastle disease, after absorption, add maintenance medium, putting 37～38 DEG C continues to cultivate, when reaching 75%, cytopathy gathers in the crops venom when above ,-15 DEG C of following preservations, and maintenance medium is the DMEM/F12 solution containing 2% hyclone;
(5) the compound inactivated vaccine preparation of newcastle disease: the virus liquid obtaining in step (4) is carried out to steriling test, viral level mensuration, carry out after the assay was approved formalin-inactivated.
The preparation of embodiment 2 herbal polysaccharides
A, press 12 parts of ginsengs, 12 parts, Poria cocos, 25 parts of plantain seeds, 12 parts of the Radixs Astragali, 15 parts, Radix Glycyrrhizae, 22 parts of pawpaws, 12 parts of gingkoes, 15 parts of reed rhizomes take each Chinese medicine material, chopping, spend the night by cold water soak after cleaning, then add the purified water of 15 times of raw material weights, water-bath to 90 DEG C, and remain on 90 DEG C, and boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect liquid, after room temperature is cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out to alcohol precipitation, precipitation is rough herbal polysaccharide;
D, with rough herbal polysaccharide in sterilizing purified water washing c, after washing, dissolves with 42 DEG C of sterilizing purified water of 20 times of amounts, after dissolving, add 4 DEG C of absorption of active carbon to spend the night in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, by supernatant in d after the degerming of 0.22um membrane filtration, carry out 10 times concentrated, make herbal polysaccharide concentrate;
F, herbal polysaccharide concentrate in e is carried out to vacuum freeze drying obtain herbal polysaccharide.
Also can be by 10 parts of ginsengs in this embodiment, 10 parts, Poria cocos, 20 parts of plantain seeds, 10 parts of the Radixs Astragali, 10 parts, Radix Glycyrrhizae, 20 parts of pawpaws, 10 parts of gingkoes, 10 parts of reed rhizomes take each Chinese medicine material; Or press 30 parts of ginsengs, and 30 parts, Poria cocos, 40 parts of plantain seeds, 20 parts of the Radixs Astragali, 30 parts, Radix Glycyrrhizae, 30 parts of pawpaws, 20 parts of gingkoes, 30 parts of reed rhizomes take each Chinese medicine material.
The preparation of embodiment 3 bursopoietins
A, get Jian Kangfashi lens capsule tissue, bursa of farbricius tissue is rejected to manadesma and adipose tissue, the cold PBS of pH7.2 sterilizing cleans.
B, weighing bursa of farbricius weight, in 1:1(W:V) ratio adds the cold PBS of pH7.2 sterilizing, carries out high-speed homogenization in tissue refiner.
In c, homogenate, add 2.5% trypsase, multigelation 3 times, the centrifugal 20min of 12000rpm, abandons precipitation.
D, supernatant carry out ultrafiltration with the milipore filter of 1000da molecular cut off, and under film, liquid is bursopoietin crude extract.
E, by crude extract in d by the degerming of 0.22um membrane filtration, then obtain bursopoietin through vacuum freezedrying.
The preparation of embodiment 4 newcastle disease Yolk antibodies
A, use newcastle disease efficient concentration inactivated vaccine immune health laying hen. Immune programme for children is 0.5ml/ of chest muscle immunity first; Head exempts to carry out secondary immunity after one week, and chest muscle immunity 1.0ml/ only; Within after secondary immunity two weeks, carry out three immunity, chest muscle immunity 1.0ml/ only.
After b, three immunity, within two weeks, start to monitor high-immunity egg antibody titer, in the time that newcastle disease, the diffusion of avian influenza virus Yolk antibody agar are tired and reached 1:128 in high-immunity egg, gather qualified high-immunity egg.
C, yolk separate: by b, gather without 0.1% bromogeramine cleaning and sterilizing damaged high-immunity egg for, broken shell separates yolk, collection yolk, in yolk storage tank, stirs and evenly mixs.
D, acidifying water extraction: by 1:11, purified water is added in acidifying extractor, yolk in c is injected to acidifying extractor, stir, measure yolk diluent pH, with 1M watery hydrochloric acid or 1M NaOH adjusting pH to 6.0,4 DEG C of static hatching 24 hours.
E, ultrafiltration concentration: draw supernatant in d, through continuous flow centrifuge, 10000rpm is centrifugal, centrifugal rear supernatant carries out 60-80 through 30kd Mi Libo ultrafiltration concentration device and doubly concentrates.
F, Yolk antibody concentrate in e is made to Yolk antibody freeze-dried powder through vacuum freezedrying.
Embodiment 5 immunopotentiator preparations
Take wherein pyridoxamine 2.4g of herbal polysaccharide 20g, vitamin combination 10g(, folic acid 6.2g, vitamin C 1.4g), bursopoietin 15mg, Yolk antibody 10g; Above-mentioned each component is mixed as portion metering and used. Wherein herbal polysaccharide is by making according to the step of embodiment 2, bursopoietin according to the step of embodiment 3 make, Yolk antibody makes according to the step of embodiment 4.
Embodiment 6 immunopotentiator preparations
Take wherein pyridoxamine 2.0g of herbal polysaccharide 30g, vitamin combination 8g(, folic acid 4.2g, vitamin C 1.8g), bursopoietin 12mg, Yolk antibody 5g; Above-mentioned each component is mixed as portion metering and used. Wherein herbal polysaccharide is by making according to the step of embodiment 2, bursopoietin according to the step of embodiment 3 make, Yolk antibody makes according to the step of embodiment 4.
Embodiment 7 immunopotentiator preparations
Take wherein pyridoxamine 0.9g of herbal polysaccharide 50g, vitamin combination 6g(, folic acid 3.3g, vitamin C 1.8g), bursopoietin 2mg, Yolk antibody 25g; Above-mentioned each component is mixed as portion metering and used. Wherein herbal polysaccharide is by making according to the step of embodiment 2, bursopoietin according to the step of embodiment 3 make, Yolk antibody makes according to the step of embodiment 4.
The preparation method of 8 one kinds of compound inactivated vaccines of newcastle disease of embodiment
Under a, aseptic condition, newcastle disease inactivation of viruses suspension liquid in embodiment 1 is added in embodiment 5,6 or 7 to 1 or 2 parts of the immunopotentiators of preparation by every 1000ml, mix, immunopotentiator is dissolved completely.
B, by the adding in emulsifier containing the newcastle disease virus inactivation antigen liquid of immunopotentiator of 3 deals, regulating emulsifier rotor speed is 1500rpm, and the Sang meter Te water profit adjuvant of 2 deals is slowly added to the antigen liquid in stirring.
C, in the time that oil phase adds, regulating rotary rotor speed is 3000rpm, continue stir 3-5 minute.
After d, emulsification completely, by aseptic subpackaged vaccine after the emulsification compound inactivated vaccine of newcastle disease of making.
F, product inspection
(1) assay
According to " People's Republic of China's veterinary drug allusion quotation " (2010 editions) related request, finished product is carried out to related check, assay is in table 1:
Table 1 product inspection result
(2) immune effect of vaccine comparison
Get 80 1 monthly age SPF chickens, be divided into two groups, experimental group and control group. The compound inactivated vaccine of experimental group immunity the present invention, the common inactivated vaccine of composite immune reinforcing agent is not added in control group immunity, and immunity plays blood sampling for latter 3 days and detects its HI antibody titer.

Claims (7)

1. a preparation method for the compound inactivated vaccine of newcastle disease, is characterized in that comprising following processing step:
(1) plant going down to posterity and cultivating of cell: chicken embryo continuous cell line, through pancreatin cell dispersion liquid had digestive transfer culture, continues to cultivate with cell growth medium, while forming good individual layer, goes down to posterity or virus inoculation for continuing;
(2) breeding of cell adapted seed culture of viruses: get cell adapted NDV seed culture of viruses, be inoculated in the chicken embryo passage cell that has grown up to individual layer in step (1), put 37～38 DEG C and continue to cultivate, cytopathy variability reaches 75% liquid of harvesting when above;
(3) large-scale culture of cell for seedling: step (1) is cultivated to the kind cell suspending liquid obtaining and be seeded in rolling bottle or cell factory, add cell growth medium to put 37～38 DEG C and cultivate;
(4) preparation of venom for seedling: get the above-mentioned clone blake bottle that has formed good individual layer in step (3), discard cell growth medium, the cell adapted malicious viral suspension of inoculation ewcastle disease, after absorption, add maintenance medium, putting 37～38 DEG C continues to cultivate, when reaching 75%, cytopathy gathers in the crops venom when above ,-15 DEG C of following preservations;
(5) the compound inactivated vaccine preparation of newcastle disease: the virus liquid obtaining in step (4) is carried out to steriling test, viral level mensuration, carry out after the assay was approved formalin-inactivated, deactivation after the assay was approved, in the Newcastle Disease Virus Antigen liquid being up to the standards in every 1000ml deactivation, add 1～2 portion of immunopotentiator to mix, then with Sang meter Te domestic animal W/O/W adjuvant by volume 3:2 carry out emulsification packing and make the compound inactivated vaccine of newcastle disease.
2. the preparation method of the compound inactivated vaccine of a kind of newcastle disease as claimed in claim 1, it is characterized in that in described step 4), every portion of immunopotentiator contains herbal polysaccharide 20～50g, vitamin combination 6～10g, bursopoietin 2～16mg and Yolk antibody 5～25g, be preferably herbal polysaccharide 30～40g, vitamin combination 7～9g, bursopoietin 8～12mg and Yolk antibody 10～20g.
3. the preparation method of the compound inactivated vaccine of a kind of newcastle disease as claimed in claim 2, is characterized in that described herbal polysaccharide makes by following steps:
A, press 10～30 parts of ginsengs, 10～30 parts, Poria cocos, 20～40 parts of plantain seeds, 10～20 parts of the Radixs Astragali, 10～30 parts, Radix Glycyrrhizae, 20～30 parts of pawpaws, 10～20 parts of gingkoes, 10～30 parts of reed rhizomes take each Chinese medicine material, chopping, spend the night by cold water soak after cleaning, then add the purified water of 15 times of raw material weights, water-bath to 90 DEG C, and remain on 90 DEG C, and boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect liquid, after room temperature is cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out to alcohol precipitation, precipitation is rough herbal polysaccharide;
D, with rough herbal polysaccharide in sterilizing purified water washing c, after washing, dissolves with 42 DEG C of sterilizing purified water of 20 times of amounts, after dissolving, add 4 DEG C of absorption of active carbon to spend the night in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, by supernatant in d after the degerming of 0.22um membrane filtration, carry out 10 times concentrated, make herbal polysaccharide concentrate;
F, herbal polysaccharide concentrate in e is carried out to vacuum freeze drying obtain herbal polysaccharide.
4. the preparation method of the compound inactivated vaccine of a kind of newcastle disease as claimed in claim 2, is characterized in that containing B615～35% of supporting one's family, folic acid 40～70% and vitamin C 10～30% in described vitamin combination.
5. the preparation method of the compound inactivated vaccine of a kind of newcastle disease as claimed in claim 2, it is characterized in that described bursopoietin makes by following steps: bursa of farbricius tissue is rejected to manadesma and adipose tissue, the cold PBS of pH7.2 sterilizing cleans, add the cold PBS of pH7.2 sterilizing in 1:1 ratio, in tissue refiner, carry out high-speed homogenization, in homogenate, add the trypsase that accounts for 2.5% weight, multigelation 3 times, the centrifugal 20min of 12000rpm, abandon precipitation, supernatant carries out ultrafiltration with the milipore filter of 1000da molecular cut off, under film, liquid is through the degerming of 0.22um membrane filtration, filtered solution is bursopoietin crude extract, crude extract obtains bursopoietin through vacuum freezedrying.
6. the preparation method of the compound inactivated vaccine of a kind of newcastle disease as claimed in claim 2, is characterized in that described Yolk antibody adopts newcastle disease efficient concentration inactivated vaccine Immune Laying Hens, separates yolk, and acidifying makes through vacuum freeze drying after extracting.
7. the preparation method of the compound inactivated vaccine of a kind of newcastle disease as claimed in claim 4, it is characterized in that in described step a by 12～26 parts of ginsengs, 12～26 parts, Poria cocos, 25～35 parts of plantain seeds, 12～18 parts of the Radixs Astragali, 15～25 parts, Radix Glycyrrhizae, 22～28 parts of pawpaws, 12～18 parts of gingkoes, 15～50 parts of reed rhizomes take each Chinese medicine material.
CN201511006408.0A 2015-12-29 2015-12-29 Preparation method of compound inactivated vaccine for newcastle disease Pending CN105597093A (en)

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Application publication date: 20160525