CN104888213A - Preparation method of classical swine fever spleen-lymph-sourced compound living vaccine - Google Patents
Preparation method of classical swine fever spleen-lymph-sourced compound living vaccine Download PDFInfo
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Abstract
The invention relates to a preparation method of a classical swine fever spleen-lymph-sourced compound living vaccine and belongs to the technical field of veterinary biological products. The preparation method of the classical swine fever spleen-lymph-sourced compound living vaccine comprises the following process steps: selecting a domestic rabbit to be inoculated; inoculating; measuring the temperature and observing; harvesting; preparing virus suspension and inspecting; carrying out mixed adsorption on the harvested full-suspension virus bulk and chicken anti-hog cholera virus egg yolk antibody; and adding freezing and drying protecting agent containing an immunopotentiator into antigen antibody compound obtained through adsorption, carrying out split charging, freezing, and carrying out vacuum drying, so that the classical swine fever spleen-lymph-sourced compound living vaccine is obtained. By adopting the preparation method of the classical swine fever spleen-lymph-sourced compound living vaccine, an antibody acquiring route is simple, animal welfare can be improved, manpower and material resources are saved, production cost is greatly reduced, humoral immunity of the living vaccine can be greatly improved, and high neutralizing antibodies can be produced; and the classical swine fever spleen-lymph-sourced compound living vaccine can be applied to piglet immunization, maternal antibody interference can be avoided, no interference is produced to other vaccines, especially a mycoplasma hyopneumoniae vaccine and the like, after the classical swine fever spleen-lymph-sourced compound living vaccine is applied to piglets, and strong mucosal immune response can be induced, so that protection rate is improved.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to the preparation method that a kind of swine fever spleen drenches source composite live vaccine.
Background technology
Swine fever (CSF) is the category-A Notifiable disease of International Animal Health tissue (OIE) and China, is one of great animal epidemic of the mandatory immunity of China, is harm China's pig industry " the first killer ".Swine fever is caused by swine fever virus, cannot control with medicine, and vaccine immunity is the most effective way.
Swine Fever Vaccine commercially available both at home and abroad mainly contains the pouring of swine fever spleen Seedling, swine fever cell vaccine and subunit vaccine for swine fever at present, but on market, the main product of swine Fever Vaccine remains swine fever spleen pouring Seedling and swine fever cell vaccine.Drench source live vaccine as the classics in vaccine-refining swine fever spleen and still there is irreplaceable effect.
Swine fever spleen drenches source live vaccine selects Chinese fever virus lapinized Chinese Strain (C strain), and this vaccine strain energy excitating organism produces immunity, inheritance stability, virulence fast and do not return by force, is the current attenuated vaccine strain the most safely and effectively in the world of generally acknowledging in the world.Adopt this strain to inoculate excellent rabbit, the spleen of results infected rabbits makes Emulsion, makes swine fever spleen drench source live vaccine with freeze drying protectant after mixing through lyophilisation.
It is undesirable that existing market thinks that swine fever spleen drenches source live vaccine immune effect, still there are mistaken ideas in the understanding of swine fever spleen being drenched to live vaccine, this main and current domestic swine fever spleen drenches that source live vaccine using method is improper, stress be excessive after the immunity of mixed infection pig, and quality is uneven etc., and situation is relevant.
Polysaccharide is one of active ingredient of Chinese herbs, and large quantity research shows, 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 various activities of cell in biosis.Polysaccharose substance is the important component part of all Living organisms; there is scavenging free radicals, improve the ability of activities of antioxidant enzymes and anti-lipid peroxidation; polysaccharide also has good heat-resisting effect simultaneously, and kinds of experiments shows that herbal polysaccharide can be used as heat resisting protective and makes an addition in live vaccine freeze drying protectant.
Vitamin is requisite organic compound in organism metabolism.The chemical plant that body is very complicated just as one, constantly carries out various biochemical reaction.Its reaction has substantial connection with the catalytic action of enzyme.Enzyme will produce activity, and coenzyme must be had to participate in.Known many vitamin are the coenzyme of enzyme or the ingredient of coenzyme.Therefore, vitamin is the important substance maintaining and regulate body homergy.
Flavone compound is the polyatomic phenol material that occurring in nature exists, and is also one of main active in nature medicinal plants.It refers to have 15 carbon atoms and with the three ring natural organic matters that the mode of C6-C3-C6 is formed, be the secondary metabolism product that plant produces in long-term natural selection, now isolation identification have kind more than 4000.It is extensively present in fruit and vegerable, Chinese herbal medicine, have no side effect, it has the medicine healthy sofa functions such as significant scavenger interior free yl, aging resistance, mutation, Adjust-blood lipid blood pressure lowering, it is the natural organic oxidation-resistant agent that a class has DEVELOPMENT PROSPECT, these anti-oxidation active substances can reduce and scavenger interior free yl, have prophylactic effect.
Antigen-antibody complex vaccine is a kind of new generation vaccine grown up the nineties in last century, is mixed according to proper proportion and antigen by specificity hyper-immune serum.In antigen-antibody complex vaccine, antibody mainly plays a part to delay virus release, and after vaccine injection, the one that immune complex can be present in spleen is called that dendron shape lymphocyte caught, and antigen can slow releasing then.When maternal antibody drops to the level that vaccine can go out brokenly, the vaccine antigen now discharged can in target organ field planting, thus induced immunity.Its advantage is that safety is high, and immune effect is better than conventional vaccine; Can break through maternal antibody interference, piglet uses such vaccine to the noiseless effect of other vaccine immunities.
Traditional antigen-antibody complex vaccine manufacture method is combined with bursal disease virus by hyper-immune serum, the method prepares the required a large amount of hyper-immune serum antibody of vaccine, need obtain, wasting time and energy by massacring a large amount of animal, production cost is higher simultaneously, causes vaccine price costly.
The research of yolk antibody started from for 19 beginnings of the century in the end of the century 20.1889, Klemperer found to be rich in antibody in yolk; 1934, the antibody that the experiment of Jukes demonstrates in hen serum can be transferred in yolk, thus provided passive immune protection for chickling.
Structure and the mammalian immunoglobulin of yolk antibody (IgY) molecule are similar.Yolk antibody is as a kind of immunoglobulin with biologic activity, and in production, processing, stores and to ingest and in digestion process, and the activity keeping antibody is crucial.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 virus IgY activity.Result show IgY still keep after pepsin enzymolysis 24h in and the effect of antigen, its enzymatic fragment also has antibody activity.Yolk antibody also has unrivaled superiority in production.First, produce antigen amount needed for effective immune response little, in the mammalian proteins confrontation phylogenetics of especially high conservative, distant birds has stronger immunogenicity usually.Secondly, collect egg instant, without the need to blood sampling, not damaged, meets modem animal safeguard rule.Europe alternative method authentication center (ECVAM) suggestion replaces mammal IgG as experiment and the source producing antibody using IgY.3rd, each egg, approximately containing the IgY of more than 100mg, can reach 3g for one month and be equivalent to the 10-20 of rabbit doubly; There is no other Ig in yolk, be easy to purify.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.The IgY so can purify on a large scale again when egg is collected a certain amount of.
The present invention is based on above-mentioned technical background; propose a kind of swine fever spleen and drench source composite live vaccine and preparation method thereof; this vaccine combines by proper proportion primarily of swine fever virus and chicken anti-Porcine reproductive and respiratory syndrome swine fever virus yolk antibody, and polysaccharide flavone complex freeze drying protectant is made through vacuum lyophilization.Prepare antigen-antibody complex vaccine by the method, antibody access approaches is simple, can improve animal welfare, use manpower and material resources sparingly, significantly reduce production cost, greatly can improve the humoral immunization of live vaccine, produce higher neutralizing antibody.This vaccine may be used for piglet immunological, can break through maternal antibody interference, and piglet is noiseless to other vaccines after using this vaccine, particularly i (mycoplasma hyopneumoniae) vaccine etc., the mucosal immune response simultaneously generation can being induced stronger, improves protective rate.
Summary of the invention
For prior art Problems existing; the object of the invention is to design the technical scheme providing a kind of swine fever spleen to drench the preparation method of source composite live vaccine; this freeze drying protectant can increase substantially the immunity of body; improve humoral immunization ability; stimulate body to produce efficient neutralizing antibody, improve the prophylactic ability of body.
Described a kind of swine fever spleen drenches the preparation method of source composite live vaccine, it is characterized in that comprising following processing step:
(1) selection of rabbit is inoculated
Growth selection in order, the rabbit of body weight 1.5-3kg;
(2) inoculate
Get production seed culture of viruses, by diluted, every family rabbit ear vein inoculation 1ml, after inoculation, rabbit is normally raised;
(3) thermometric is observed
After family's rabbit inoculation, the upper and lower noon respectively surveys body temperature once, after 24 hours, surveys body temperature once, thermometric to 72 hour every 6 hours;
(4) gather in the crops
Select sizing heat and slight fever to react rabbit, kill from temperature decline and cuing open in later 24 hours, take spleen with aseptic operation;
(5) viral suspension preparation and inspection
Under sterile working, reject fatty tissue, weighed by spleen tissue, shred, mixed grinding in same container, then filters, and measures viral level, minimal infecting dose (MID) Ying≤10 of spleen poison to rabbit
5/ ml;
(6) the complete outstanding virus liquid of above-mentioned results and chicken swine fever virus resistant yolk antibody are carried out mixing and absorption;
(7) antigen antibody complex after absorption is added containing immunostimulant freeze drying protectant, make pig swine fever spleen pouring source through lyophilisation after subpackage and levy composite live vaccine.
Described a kind of swine fever spleen drenches the preparation method of source composite live vaccine, to it is characterized in that in described step (6) complete outstanding virus liquid and chicken swine fever virus resistant yolk antibody by volume 1:1 mix, described complete outstanding virus liquid every milliliter antigen contains viral 10
5rID, described chicken swine fever virus resistant yolk antibody often milli is tired as 1:1024.
Described a kind of swine fever spleen drenches the preparation method of source composite live vaccine, it is characterized in that in described step (6), the 15 DEG C of mixing of mixing and absorption condition hatch 2 hours.
Described a kind of swine fever spleen drenches the preparation method of source composite live vaccine, to it is characterized in that in described step (7) antigen antibody complex and freeze drying protectant by volume 1:1 mix.
Described a kind of swine fever spleen drenches the preparation method of source composite live vaccine; it is characterized in that in described step (7), freeze drying protectant is containing herbal polysaccharide 30 ~ 100g, Chinese medicine flavone 20 ~ 60g, vitamin combination 5 ~ 20g in every 1000ml phosphate buffer; described herbal polysaccharide and Chinese medicine flavone extract and obtain from following Chinese medicine composition: Herba Sedi, Radix Tinosporae, fevervine, Cornu Bubali, Gryllotalpa, Herba Moslae, Radix Stephaniae Tetrandrae, Herba Lobeliae Chinensis and Fructus Liquidambaris, and described vitamin combination is containing folic acid, nicotiamide, vitamin B and vitamin E.
Described a kind of swine fever spleen drenches the preparation method of source composite live vaccine, it is characterized in that the extraction step of described herbal polysaccharide and Chinese medicine flavone is:
A, take each raw material of Chinese medicine by Herba Sedi 10 ~ 30 parts, Radix Tinosporae 20 ~ 40 parts, fevervine 10 ~ 20 parts, Cornu Bubali 10 ~ 30 parts, Gryllotalpa 10 ~ 30 parts, Herba Moslae 10 ~ 20 parts, Radix Stephaniae Tetrandrae 10 ~ 30 parts, Herba Lobeliae Chinensis 10 ~ 30 parts and Fructus Liquidambaris 10 ~ 20 parts, chopping, clean after spend the night by cold water soak, add the purified water of raw material weight 15 times again, water-bath to 90 DEG C, and remain on 90 DEG C, boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect medicinal liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out precipitate with ethanol, precipitation is rough herbal polysaccharide, and supernatant is rough Chinese medicine flavone;
D, with rough herbal polysaccharide in sterilizing purified water washing c, dissolves with 42 DEG C of sterilizing purified water of 20 times amount after washing, after dissolving, carry out Endotoxin adsorption by by activated-charcoal column, collection filter liquor;
E, will in d upper filter liquor by sevag method except Deproteinization;
F, herbal polysaccharide in e is carried out vacuum lyophilization obtain herbal polysaccharide;
G, Chinese medicine flavone rough in c is carried out reduced vacuum concentrate, concentrate sterilized water for injection dilution cleaning, again carries out reduced vacuum and concentrates, make Chinese medicine flavone after three times like this through lyophilisation.
Described a kind of swine fever spleen drenches the preparation method of source composite live vaccine, it is characterized in that described vitamin combination is containing folic acid 20 ~ 30%, nicotiamide 30 ~ 50% vitaminB10 ~ 30% and vitamin E 20 ~ 60%.
The present invention has following beneficial effect:
1. the antigen-antibody complex vaccine prepared of the present invention, greatly can improve the humoral immunization of live vaccine, produce higher neutralizing antibody.This vaccine may be used for piglet immunological, can break through maternal antibody interference, and piglet is noiseless to other vaccines after using this vaccine, the mucosal immune response simultaneously generation can being induced stronger, improves protective rate.
2. yolk antibody instead of serum antibody and prepares antigen-antibody complex vaccine, and antibody access approaches is simple, can improve animal welfare, use manpower and material resources sparingly, significantly reduce production cost.
3. present invention employs effective immunostimulant, increased substantially the immunity of body, improve humoral immunization ability, stimulate body to produce efficient neutralizing antibody, improve 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, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention and place restrictions on scope of the present invention, NM specific experiment method in the following example, experimental technique carries out routinely usually.
Embodiment 1: the preparation of herbal polysaccharide and Chinese medicine flavone
A, take each raw material of Chinese medicine by Herba Sedi 30 parts, Radix Tinosporae 20 parts, fevervine 20 parts, Cornu Bubali 30 parts, Gryllotalpa 30 parts, Herba Moslae 10 parts, Radix Stephaniae Tetrandrae 10 parts, Herba Lobeliae Chinensis 10 parts and Fructus Liquidambaris 10 parts, chopping, clean after spend the night by cold water soak, add the purified water of raw material weight 15 times again, water-bath to 90 DEG C, and remain on 90 DEG C, boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect medicinal liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out precipitate with ethanol, precipitation is rough herbal polysaccharide, and supernatant is rough Chinese medicine flavone;
D, with rough herbal polysaccharide in sterilizing purified water washing c, dissolves with 42 DEG C of sterilizing purified water of 20 times amount after washing, after dissolving, add active carbon 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, by supernatant in d after 0.22um membrane filtration is degerming, carry out 10 times and concentrate, obtained herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out vacuum lyophilization namely obtain herbal polysaccharide;
G, Chinese medicine flavone rough in c is carried out reduced vacuum concentrate, concentrate sterilized water for injection dilution cleaning, again carries out reduced vacuum and concentrates, after three times like this, more degerming through 0.22um membrane filtration, then namely obtain Chinese medicine flavone through lyophilisation.
Herba Sedi 10 parts, Radix Tinosporae 30 parts, fevervine 15 parts, Cornu Bubali 20 parts, Gryllotalpa 20 parts, Herba Moslae 20 parts, Radix Stephaniae Tetrandrae 20 parts, Herba Lobeliae Chinensis 20 parts and Fructus Liquidambaris 20 parts can be taken in this embodiment in step a process, also can take and process by Herba Sedi 15 parts, Radix Tinosporae 40 parts, fevervine 15 parts, Cornu Bubali 15 parts, Gryllotalpa 25 parts, Herba Moslae 15 parts, Radix Stephaniae Tetrandrae 15 parts, Herba Lobeliae Chinensis 25 parts and Fructus Liquidambaris 15 parts.
The preparation of embodiment 2 chicken swine fever virus resistant yolk antibody, comprises the following steps:
A, use CSF dead vaccine immune health laying hen.Immune programme for children is chest muscle immunity first 0.5ml/; 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.
Within two weeks, start to monitor high-immunity egg antibody titer after b, three immunity, when porcine reproductive and respiratory syndrome virus yolk antibody agar diffusion anti-in high-immunity egg tire reach 1:64 time, gather qualified high-immunity egg.
C, yolk be separated: by gather in b without damaged high-immunity egg 0.1% bromo geramine cleaning and sterilizing, broken shell be separated yolk, collection yolk in yolk storage tank, stirring and evenly mixing.
D, acidify water extraction: add in acidify extraction pot by purified water by 1:11, inject acidify extraction pot by yolk in c, stir, and measures yolk diluent pH, regulates pH to 6.0 with 1M dilute hydrochloric acid or 1M sodium hydroxide, and 4 DEG C static hatches 24 hours.
E, ultrafiltration concentration: draw supernatant in d, centrifugal through continuous flow centrifuge 10000rpm, centrifugal rear supernatant carries out 60-80 through 30kd Mi Libo ultrafiltration and concentration device and doubly concentrates.
F, yolk antibody concentrated solution in e is made yolk antibody lyophilized powder through lyophilisation.
Embodiment 3: a kind of swine fever spleen drenches the preparation of source composite live vaccine freeze drying protectant
Herbal polysaccharide 60g, Chinese medicine flavone 40g, vitamin combination 10g(folic acid 2g, nicotiamide 3g, vitamin B 1g and vitamin E 4g is added in every 1000ml phosphate buffer), namely obtain swine fever spleen and drench source live vaccine freeze drying protectant.Wherein herbal polysaccharide and Chinese medicine flavone obtain according to the step in embodiment 1.Phosphate buffer by sodium chloride 6 ~ 10g, potassium chloride 0.05 ~ 0.5g, sodium hydrogen phosphate 1 ~ 1.2g, potassium dihydrogen phosphate 0.05 ~ 0.5g, calcium chloride 0.05 ~ 0.2g, the magnesium chloride 0.05 ~ 0.2g containing 6 water of crystallization is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving obtains for 30 minutes.
Embodiment 4: a kind of swine fever spleen drenches the preparation of source composite live vaccine freeze drying protectant
Herbal polysaccharide 100g, Chinese medicine flavone 60g, vitamin combination 16g(folic acid 4g, nicotiamide 5g, vitamin B 3g and vitamin E 4g is added in every 1000ml phosphate buffer), namely obtain swine fever spleen and drench source live vaccine freeze drying protectant.Wherein herbal polysaccharide and Chinese medicine flavone obtain according to the step in embodiment 1.Phosphate buffer by sodium chloride 6 ~ 10g, potassium chloride 0.05 ~ 0.5g, sodium hydrogen phosphate 1 ~ 1.2g, potassium dihydrogen phosphate 0.05 ~ 0.5g, calcium chloride 0.05 ~ 0.2g, the magnesium chloride 0.05 ~ 0.2g containing 6 water of crystallization is dissolved in 1000ml distilled water, through 116 DEG C, autoclaving obtains for 30 minutes.
Embodiment 5: a kind of swine fever spleen drenches the preparation method of source composite live vaccine, comprises the following steps:
(1) selection of rabbit is inoculated
Growth selection in order, the rabbit 100 of body weight 1.5-3kg.
(2) inoculate
Get production seed culture of viruses, do 10 with diluent
-1.5doubly dilution, every family rabbit ear vein inoculation 1ml, after inoculation, rabbit is normally raised.
(3) thermometric is observed
After family's rabbit inoculation, the upper and lower noon respectively surveys body temperature once, after 24 hours, surveys body temperature once every 6 hours.Thermometric to 72 hour.
(4) gather in the crops
Select sizing heat and slight fever to react rabbit, kill from temperature decline and cuing open in later 24 hours, take spleen with aseptic operation.
(5) viral suspension preparation and inspection
Fatty tissue is rejected under sterile working.Weighed by spleen tissue, shred, mixed grinding in same container, then filters, and measures viral level, minimal infecting dose (MID) Ying≤10 of spleen poison to rabbit
5/ ml.
(6) by the complete outstanding virus liquid of above-mentioned results, (complete outstanding virus liquid every milliliter antigen is containing virus 10
5rID) at 15 DEG C, mixing and absorption is carried out 2 hours with the chicken swine fever virus resistant yolk antibody of embodiment 2 by 1:1.
(7) by the antigen antibody complex after absorption in 1:1(V:V) ratio adds the obtained freeze drying protectant of embodiment 3 or 4, makes swine fever spleen pouring source composite live vaccine (experimental group) after subpackage through lyophilisation.
(8) by the complete outstanding virus liquid in step (5) with sucrose milk freeze drying protectant in 1:1(V:V) ratio mixs homogeneously, and makes swine fever spleen pouring source live vaccine (matched group) after subpackage through lyophilisation.
(9) vaccine test
1) with the holding time, indices assay
Two of above-mentioned preparation batches of vaccines are placed room temperature (25 DEG C) preserve, and carry out viral level detection every other month, testing result is in table 1:
Table 1 storage life testing result (25 DEG C)
2) immune effect of vaccine
Get 20 hog cholera antibody negative healthy piglets, be divided into two groups, experimental group and matched group.Experimental group immunity is containing frozen-dried protective vaccinating agent of the present invention, and matched group immunity is containing sucrose milk freeze drying protectant vaccine, and immunity plays blood sampling for latter 3 days and detects its NAT, the results are shown in Table 2
Table 2 immunological comparison result
Claims (7)
1. swine fever spleen drenches a preparation method for source composite live vaccine, it is characterized in that comprising following processing step:
(1) selection of rabbit is inoculated
Growth selection in order, the rabbit of body weight 1.5-3kg;
(2) inoculate
Get production seed culture of viruses, by diluted, every family rabbit ear vein inoculation 1ml, after inoculation, rabbit is normally raised;
(3) thermometric is observed
After family's rabbit inoculation, the upper and lower noon respectively surveys body temperature once, after 24 hours, surveys body temperature once, thermometric to 72 hour every 6 hours;
(4) gather in the crops
Select sizing heat and slight fever to react rabbit, kill from temperature decline and cuing open in later 24 hours, take spleen with aseptic operation;
(5) viral suspension preparation and inspection
Under sterile working, reject fatty tissue, weighed by spleen tissue, shred, mixed grinding in same container, then filters, and measures viral level, minimal infecting dose (MID) Ying≤10 of spleen poison to rabbit
5/ ml;
(6) the complete outstanding virus liquid of above-mentioned results and chicken swine fever virus resistant yolk antibody are carried out mixing and absorption;
(7) antigen antibody complex after absorption is added containing immunostimulant freeze drying protectant, make pig swine fever spleen pouring source through lyophilisation after subpackage and levy composite live vaccine.
2. a kind of swine fever spleen as claimed in claim 1 drenches the preparation method of source composite live vaccine, to it is characterized in that in described step (6) complete outstanding virus liquid and chicken swine fever virus resistant yolk antibody by volume 1:1 mix, described complete outstanding virus liquid every milliliter antigen contains viral 10
5rID, described chicken swine fever virus resistant yolk antibody often milli is tired as 1:1024.
3. a kind of swine fever spleen as claimed in claim 1 drenches the preparation method of source composite live vaccine, it is characterized in that in described step (6), the 15 DEG C of mixing of mixing and absorption condition hatch 2 hours.
4. a kind of swine fever spleen as claimed in claim 1 drenches the preparation method of source composite live vaccine, to it is characterized in that in described step (7) antigen antibody complex and freeze drying protectant by volume 1:1 mix.
5. a kind of swine fever spleen as claimed in claim 1 drenches the preparation method of source composite live vaccine; it is characterized in that in described step (7), freeze drying protectant is containing herbal polysaccharide 30 ~ 100g, Chinese medicine flavone 20 ~ 60g, vitamin combination 5 ~ 20g in every 1000ml phosphate buffer; described herbal polysaccharide and Chinese medicine flavone extract and obtain from following Chinese medicine composition: Herba Sedi, Radix Tinosporae, fevervine, Cornu Bubali, Gryllotalpa, Herba Moslae, Radix Stephaniae Tetrandrae, Herba Lobeliae Chinensis and Fructus Liquidambaris, and described vitamin combination is containing folic acid, nicotiamide, vitamin B and vitamin E.
6. a kind of swine fever spleen as claimed in claim 5 drenches the preparation method of source composite live vaccine, it is characterized in that the extraction step of described herbal polysaccharide and Chinese medicine flavone is:
A, take each raw material of Chinese medicine by Herba Sedi 10 ~ 30 parts, Radix Tinosporae 20 ~ 40 parts, fevervine 10 ~ 20 parts, Cornu Bubali 10 ~ 30 parts, Gryllotalpa 10 ~ 30 parts, Herba Moslae 10 ~ 20 parts, Radix Stephaniae Tetrandrae 10 ~ 30 parts, Herba Lobeliae Chinensis 10 ~ 30 parts and Fructus Liquidambaris 10 ~ 20 parts, chopping, clean after spend the night by cold water soak, add the purified water of raw material weight 15 times again, water-bath to 90 DEG C, and remain on 90 DEG C, boil 2 hours, boil in process every 10 minutes and stir once;
B, discard a Chinese medicine slag, collect medicinal liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out precipitate with ethanol, precipitation is rough herbal polysaccharide, and supernatant is rough Chinese medicine flavone;
D, with rough herbal polysaccharide in sterilizing purified water washing c, dissolves with 42 DEG C of sterilizing purified water of 20 times amount after washing, after dissolving, carry out Endotoxin adsorption by by activated-charcoal column, collection filter liquor;
E, will in d upper filter liquor by sevag method except Deproteinization;
F, herbal polysaccharide in e is carried out vacuum lyophilization obtain herbal polysaccharide;
G, Chinese medicine flavone rough in c is carried out reduced vacuum concentrate, concentrate sterilized water for injection dilution cleaning, again carries out reduced vacuum and concentrates, make Chinese medicine flavone after three times like this through lyophilisation.
7. a kind of swine fever spleen as claimed in claim 5 drenches the preparation method of source composite live vaccine, it is characterized in that described vitamin combination is containing folic acid 20 ~ 30%, nicotiamide 30 ~ 50% vitaminB10 ~ 30% and vitamin E 20 ~ 60%.
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| CN113166234A (en) * | 2019-11-18 | 2021-07-23 | （株）Ad生物技术 | Preparation method of yolk antibody for resisting helicobacter pylori |
| CN111202029A (en) * | 2020-02-26 | 2020-05-29 | 金宇保灵生物药品有限公司 | Establishment method and application of Seneca virus rabbit animal model |
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