CN105505888A - Infectious bursa disease virus diluent and preparation method thereof - Google Patents

Infectious bursa disease virus diluent and preparation method thereof Download PDF

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CN105505888A
CN105505888A CN201511006412.7A CN201511006412A CN105505888A CN 105505888 A CN105505888 A CN 105505888A CN 201511006412 A CN201511006412 A CN 201511006412A CN 105505888 A CN105505888 A CN 105505888A
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vitamin
polysaccharide
diluent
combination
herbal
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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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    • C12N2720/10051—Methods of production or purification of viral material

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Abstract

The invention provides an infectious bursa disease virus diluent and a preparation method thereof, and belongs to the technical field of animal biological products. The diluent is prepared from, by weight, 1-2 g of an amino acid composition, 1-3 g of a vitamin composition, 10-20 mg of sodium selenite and 10-20 g of compound Chinese medicinal herb polysaccharide in every 1000 ml of a phosphate buffer solution, wherein the amino acid composition is prepared from cystine, valine and isoleucine, the vitamin composition is prepared from vitamin B1, vitamin C and vitamin D, and the compound Chinese medicinal herb polysaccharide is prepared by extracting flos inulae, semen brassicae, rhizoma typhonii and herba lycopi. After the prepared diluent is inoculated to chick embryos, the virus resisting capacity of the chick embryos can be improved, the death time is prolonged, that is, the virus multiplication time is prolonged, and compared with the prior art, the virus titer is improved by 5 times or above.

Description

A kind of infections chicken cloacal bursa virus diluent and preparation method thereof
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of infections chicken cloacal bursa virus diluent and preparation method thereof.
Background technology
Infectious bursal disease (Infectionbursaldisease, IBD) is caused by infectious bursa of Fabricius virus (Infectionbursaldiseasevirus, IBDV) that the one endangering young chicken is acute, high degree in contact sexually transmitted disease.IBDV main infection chicken and turkey, easily betide 3-12 age in week, this virus in the lymphocyte internal breeding of the fabricius bursa, and causes damage in various degree to other immune organ, finally can cause the fabricius bursa atrophy of chicken and cause immunosuppression.Infectious bursal disease occurs suddenly, without significantly seasonal, propagate very fast, propagate usually through approach such as contaminated feed, drinking-water, ight soil, hen house apparatus, staff garments, latent period is 1-5 days, and after there is symptom the 3rd day starts dead, it within 4-6 days, is peak mortality phases, stop death gradually, sickness rate can reach more than 90%, and mortality ratio is up to 10%-50% later.This disease show as clinically lose weight, poor growth and the symptom such as skeletal muscle is hemorrhage, often occur together when sanitary condition is poor other diseases, mortality ratio improves further, and chicken individuals is little, and disease resistance is poor, once generation epidemic disease, then infect fast, mortality ratio is high, and mortality ratio reaches more than 80%, loss is larger, has carried out huge financial loss to poultry industrial belt.In addition, also can reduce the chicken group after morbidity to newcastle disease vaccine, Mareks disease vaccine immune effect, can with colibacillosis, newcastle disease, the sick polyinfection of mycoplasma gallinarum, mortality ratio can improve greatly.IBDV is very stable in external environment, can in hen house long-term existence, heat-resisting especially, 60 DEG C of 90min viruses are not inactivated; The strain type of IBDV is also in continuous variation, and the low virulent strain before the eighties becomes highly virulent strain and variation strain, thus makes classical vaccine can not resist the attack of variation strain.Along with the rise of intensive aviculture and the increase of international trade, poultry diease is popular to be on the rise, and causes the high mortality of poultry, seriously governs the development of poultry husbandry, for world's poultry husbandry causes serious loss, jeopardizes Economic development, food safety and social stability.
The infections chicken cloacal bursa living vaccine that Present Domestic is produced is substantially all breed after adopting egg inoculation virus, and results infect fetus and are prepared from through tissue grinder.That gathers in the crops because of infections chicken cloacal bursa virus is organized as fetus, and during results, fetation situation has a strong impact on semi-manufactured goods quality, and grew slow fetus less, output is lower; Fetation comparatively fast then there will be more feather tissue, reduces semi-manufactured goods quality.
After the present invention adopts unique viral dilution liquid that inoculation seed culture of viruses is carried out dilution process, inoculate to low day instar chicken embryo, infect results infection idiosome after fetus.Solve the problem of embryo development after infections chicken cloacal bursa virus inoculation fetus.
Summary of the invention
For prior art Problems existing, the object of the invention is to design the technical scheme that a kind of infections chicken cloacal bursa virus diluent and preparation method thereof is provided.
Described a kind of infections chicken cloacal bursa virus diluent, it is characterized in that the component containing following weight proportion in every 1000ml phosphoric acid buffer: combination of amino acids 1 ~ 2g, VITAMIN combination 1 ~ 3g, Sodium Selenite 10 ~ 20mg and herbal mixture polysaccharide 10 ~ 20g, described combination of amino acids comprises Gelucystine, α-amino-isovaleric acid and Isoleucine, described VITAMIN combination comprises VITMAIN B1, vitamins C and vitamins D, and described herbal mixture polysaccharide is extracted by Flos Inulae, White Mustard Seed, Rhizoma Typhonii and Herba Lycopi and obtains.
Described a kind of infections chicken cloacal bursa virus diluent, is characterized in that the pH of this diluent is 6.2 ~ 7.2.
Described a kind of infections chicken cloacal bursa virus diluent, it is characterized in that described phosphoric acid buffer is by sodium-chlor 6 ~ 10g, Repone K 0.05 ~ 0.5g, Sodium phosphate dibasic 1 ~ 1.2g, potassium primary phosphate 0.05 ~ 0.5g, calcium chloride 0.05 ~ 0.2g and being dissolved in 1000ml distilled water containing the magnesium chloride 0.05 ~ 0.2g of 6 crystal water obtains.
Described a kind of infections chicken cloacal bursa virus diluent, is characterized in that the component containing following weight proportion in described every 1000ml phosphoric acid buffer: combination of amino acids 1.2 ~ 1.6g, VITAMIN combination 1.5 ~ 2.5g, Sodium Selenite 14 ~ 16mg and herbal mixture polysaccharide 14 ~ 16g.
Described a kind of infections chicken cloacal bursa virus diluent, it is characterized in that described combination of amino acids contains the amino acid of following weight percent content: Gelucystine 20 ~ 50%, α-amino-isovaleric acid 20 ~ 40% and Isoleucine 30 ~ 60%, be preferably Gelucystine 20 ~ 35%, α-amino-isovaleric acid 25 ~ 35% and Isoleucine propylhomoserin 40 ~ 50%.
Described a kind of infections chicken cloacal bursa virus diluent, it is characterized in that the described VITAMIN of VITAMIN combination containing following weight percent content: VB11 5 ~ 40%, Catergen 0 ~ 50% and vitamin D2 0 ~ 40%, be preferably vitamin B12 0 ~ 35%, vitamins C 30 ~ 45% and Vitamin D3 500,000 I.U/GM 0 ~ 35%.
Described a kind of infections chicken cloacal bursa virus diluent, is characterized in that described herbal mixture polysaccharide obtains according to the following steps:
A, take each Chinese medicine material by Flos Inulae 20 ~ 40 parts, White Mustard Seed 5 ~ 20 parts, Rhizoma Typhonii 10 ~ 30 parts, Herba Lycopi 5 ~ 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 liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out alcohol precipitation, precipitation obtains rough herbal polysaccharide;
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 gac 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, supernatant liquor in d carried out 10 times and concentrate, obtained herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out vacuum lyophilization obtain compound Chinese medicine polysaccharide frozen dried powder.
Described a kind of infections chicken cloacal bursa virus diluent preparation method, is characterized in that comprising following processing step:
1) preparation of phosphate buffer solution
By sodium-chlor 6 ~ 10g, Repone K 0.05 ~ 0.5g, Sodium phosphate dibasic 1 ~ 1.2g, potassium primary phosphate 0.05 ~ 0.5g, calcium chloride 0.05 ~ 0.2g and be dissolved in 1000ml distilled water containing the magnesium chloride 0.05 ~ 0.2g of 6 crystal water, by 0.22 μm of filter membrane under aseptic condition, filtration sterilization is for subsequent use;
2) preparation of herbal mixture polysaccharide
A, take each raw material by Flos Inulae 20 ~ 40 parts, White Mustard Seed 5 ~ 20 parts, Rhizoma Typhonii 10 ~ 30 parts, Herba Lycopi 5 ~ 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 liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out alcohol precipitation, precipitation obtains rough herbal polysaccharide;
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 gac 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, supernatant liquor in d carried out 10 times and concentrate, obtained herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out vacuum lyophilization obtain compound Chinese medicine polysaccharide frozen dried powder;
3) inoculated into chick embryo viral dilution liquid is prepared: by combination of amino acids 1 ~ 2g, VITAMIN combination 1 ~ 3g, Sodium Selenite 10 ~ 20mg and herbal mixture polysaccharide 10 ~ 20g, be dissolved in the obtained phosphate buffer solution of step 1), abundant mixing, regulate between pH to 6.2 ~ 7.2 with acid or alkali, by 0.22 μm of filter membrane under aseptic condition, rearmounted 4 DEG C of filtration sterilization saves backup.
Described a kind of infections chicken cloacal bursa virus diluent preparation method, it is characterized in that in described step 3), combination of amino acids contains the amino acid of following weight percent content: Gelucystine 20 ~ 50%, α-amino-isovaleric acid 20 ~ 40% and Isoleucine 30 ~ 60%, be preferably Gelucystine 20 ~ 35%, α-amino-isovaleric acid 25 ~ 35% and Isoleucine 40 ~ 50%.
Described a kind of infections chicken cloacal bursa virus diluent preparation method, it is characterized in that the VITAMIN of VITAMIN combination containing following weight percent content in described step 3): VB11 5 ~ 40%, Catergen 0 ~ 50% and vitamin D2 0 ~ 40%, be preferably vitamin B12 0 ~ 35%, vitamins C 30 ~ 45% and Vitamin D3 500,000 I.U/GM 0 ~ 35%.
The invention has the beneficial effects as follows:
(1) diluent prepared by the present invention, can be supplied to the required nutrition of chicken embryo when inoculated into chick embryo simultaneously, can solve the low age in days of chicken embryo and cultivate a difficult problem.
(2) diluent prepared by the present invention, can inoculate low day instar chicken embryo, because low age in days chicken embryo tissue differentiation degree is lower, compared with prior art, virus is easier breeds in chicken embryo tissue.
(3), after the diluent inoculated into chick embryo prepared by the present invention, the ability of chicken embryo tolerance virus can be improved, extend the death time, namely extend the virus multiplication time, compared with prior art improve virus titer more than 5 times.
Embodiment
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 to limit the scope of the invention.
Embodiment 1
A kind of infections chicken cloacal bursa virus diluent, contain in the every 1000ml phosphoric acid buffer of this viral dilution liquid: combination of amino acids 1g(is Gelucystine 0.2g, α-amino-isovaleric acid 0.4g, Isoleucine 0.4g wherein), VITAMIN combination 2g(wherein vitaminB10 .5g, vitamins C 0.9g, vitamins D 0.6g), herbal mixture polysaccharide 12g, Sodium Selenite 15mg, this diluent pH is 6.5.
A preparation method for infections chicken cloacal bursa virus diluent, the steps include:
(1) preparation of phosphate buffer solution: accurately take sodium-chlor 8g by above-mentioned phosphate buffered liquid formula, Repone K 0.2g, Sodium phosphate dibasic 1.15g, potassium primary phosphate 0.2g, calcium chloride 0.1g, magnesium chloride 0.1g containing 6 crystal water is dissolved in 1000ml distilled water, and by 0.22 μm of filter membrane under aseptic condition, filtration sterilization is for subsequent use.
(2) preparation of herbal mixture polysaccharide
A, take described Chinese medicine material by Flos Inulae 30 parts, White Mustard Seed 15 parts, Rhizoma Typhonii 20 parts and Herba Lycopi 15 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 liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant.
C, adopt known alcohol deposition method, supernatant in b is carried out alcohol precipitation, is precipitated as rough herbal polysaccharide.
D, with rough 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 gac 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant.
E, adopt known vacuum-concentrcted method, supernatant liquor in d is carried out 10 times and concentrate, obtained herbal polysaccharide concentrated solution.
F, adopt known vacuum freeze-drying method, e herbal polysaccharide concentrated solution is carried out vacuum lyophilization and obtains compound Chinese medicine polysaccharide frozen dried powder.
(3) inoculated into chick embryo viral dilution liquid preparation: accurately take Gelucystine 0.2g, α-amino-isovaleric acid 0.4g, Isoleucine 0.4g, vitaminB10 .5g, vitamins C 0.9g, vitamins D 0.6g, herbal mixture polysaccharide 12g, Sodium Selenite 15mg by above-mentioned preferred diluent number of components, be dissolved in the 1000ml phosphate buffer solution in step (1) in proportion, abundant mixing, pH to 6.5 is regulated with acid or alkali, by 0.22 μm of filter membrane under aseptic condition, rearmounted 4 DEG C of filtration sterilization saves backup.
Embodiment 2
A kind of infections chicken cloacal bursa virus diluent, (this phosphoric acid buffer is by sodium-chlor 6g for the every 1000ml phosphoric acid buffer of this viral dilution liquid, Repone K 0.05g, Sodium phosphate dibasic 1g, potassium primary phosphate 0.05g, calcium chloride 0.05g, be dissolved in 1000ml distilled water containing the magnesium chloride 0.05g of 6 crystal water and obtain) in contain: combination of amino acids 1.5g(is Gelucystine 0.75g wherein, α-amino-isovaleric acid 0.3g, Isoleucine 0.45g), VITAMIN combination 1.5g(wherein vitaminB10 .3g, vitamins C 0.7g, vitamins D 0.5g), herbal mixture polysaccharide 14g(is by Flos Inulae 20 parts, White Mustard Seed 5 parts, Rhizoma Typhonii 10 parts, Herba Lycopi's 5 parts of extractions obtain), Sodium Selenite 12mg, this diluent pH is 7.0.
A preparation method for infections chicken cloacal bursa virus diluent, its step is with embodiment 1.
Embodiment 3
A kind of infections chicken cloacal bursa virus diluent, (this phosphoric acid buffer is by sodium-chlor 10g for the every 1000ml phosphoric acid buffer of this viral dilution liquid, Repone K 0.5g, Sodium phosphate dibasic 1.2g, potassium primary phosphate 0.5g, calcium chloride 0.2g, be dissolved in 1000ml distilled water containing the magnesium chloride 0.2g of 6 crystal water and obtain) in contain: combination of amino acids 2g(is Gelucystine 0.7g wherein, α-amino-isovaleric acid 0.5g, Isoleucine 0.8g), VITAMIN combination 3g(wherein VB11 .2g, vitamins C 0.6g, Dry Vitamin D3 100 cws .2g), herbal mixture polysaccharide 20g(is by Flos Inulae 40 parts, White Mustard Seed 20 parts, Rhizoma Typhonii 30 parts, Herba Lycopi's 20 parts of extractions obtain), Sodium Selenite 20mg, this diluent pH is 7.2.
A preparation method for infections chicken cloacal bursa virus diluent, its step is with embodiment 1.
Embodiment 4: a kind of using method of infections chicken cloacal bursa virus diluent
1) diluent and preparation thereof, method is with embodiment 1.
2) egg inoculation of infections chicken cloacal bursa virus is bred with cultivation
(1) selection of inoculated into chick embryo
Select well-developed 9 age in days SPF chicken embryos, 1000 pieces.
(2) inoculate
Get production seed culture of viruses, carry out 100 times of dilutions with diluent of the present invention, dilute latter 4 DEG C and hatch 30 minutes, every embryo chorioallantoic membrane inoculation 0.2ml.Seal pin hole after inoculation, put 36 ~ 37 DEG C and continue to hatch, need not egg-turning.
(3) hatch and observe
After egg inoculation, per sunshine egg once, chicken embryo dead before 36 hours is discarded.After 36 hours, every 4 ~ 8 hours photograph eggs once, take out at any time, and 48-168 hour dead chicken embryo is placed in 2 ~ 8 DEG C of coolings.
(4) gather in the crops
Taken out by the cooling chicken embryo of 4 ~ 24 hours, with iodine tincture disinfection air chamber position, then remove chorion with aseptic operation, collect fetus and chorioallantoic membrane, grouping is placed in sterile chamber, after keeping sample, puts less than-10 DEG C freezen protective.While results blastochyle, check chicken embryo one by one, as fetus is corrupt, blastochyle is muddy and have the suspicious person of any pollution to be discarded.
(5) viral level measures
Steriling test: often group clip sample of tissue respectively, carries out steriling test by " steriling test or pure checked operation code ", should without bacterial growth.
Viral level measures: added in sterile saline by 1:10 by clip tissue and grind, rear sterile saline makes 10 times of serial dilutions to 10 -8, get inoculation 10 age in days SPF chicken embryo 5 in 3 each chorioallantoic membranes of acceptable diluent degree, every embryo 0.2ml, puts 36 ~ 37 DEG C and continues to hatch 7.Calculate ELD 50, every 0.2ml viral level should be not less than 10 5.0eLD 50.
Comparative example 1: adopt comparing of above-mentioned diluted virus inoculation chicken embryo and conventional diluted virus inoculation chicken embryo indices.
1) the infections chicken cloacal bursa virus diluent that obtains of Example 1.
2) conventional viral dilution liquid and preparation, i.e. physiological saline, technology is prepared routinely.
3) egg inoculation is bred with cultivation
(1) selection of inoculated into chick embryo: select well-developed 10 age in days SPF chicken embryos, 2000 pieces;
(2) inoculate: 2000 piece of 10 age in days SPF chicken embryo in this example (1) is divided into 2 groups, first group adopts viral dilution liquid of the present invention to carry out operation inoculation, second group adopts the viral dilution liquid of routine techniques preparation to carry out viral dilution and inoculation, and two groups of extension rates are identical.
(3) hatch and observe, gather in the crops, viral suspension inspection.
4) dead time of concentration and quantity after two prescription method inoculated into chick embryo, in table 1.
First group: be of the present invention group, adopt 10 age in days egg inoculations.
Second group: be routine techniques diluent group, adopt 10 age in days egg inoculations.
5) virus harvest amount and viral level thereof after two prescription method inoculated into chick embryo, in table 2.
Compare the identical test of example 1 to embodiment 2 with the 3 infections chicken cloacal bursa virus diluents obtained, finally also can reach the identical technique effect of comparative example 1, its virus harvest amount and viral level are far longer than routine techniques diluent group.

Claims (10)

1. an infections chicken cloacal bursa virus diluent, it is characterized in that the component containing following weight proportion in every 1000ml phosphoric acid buffer: combination of amino acids 1 ~ 2g, VITAMIN combination 1 ~ 3g, Sodium Selenite 10 ~ 20mg and herbal mixture polysaccharide 10 ~ 20g, described combination of amino acids comprises Gelucystine, α-amino-isovaleric acid and Isoleucine, described VITAMIN combination comprises VITMAIN B1, vitamins C and vitamins D, and described herbal mixture polysaccharide is extracted by Flos Inulae, White Mustard Seed, Rhizoma Typhonii and Herba Lycopi and obtains.
2. a kind of infections chicken cloacal bursa virus diluent as claimed in claim 1, is characterized in that the pH of this diluent is 6.2 ~ 7.2.
3. a kind of infections chicken cloacal bursa virus diluent as claimed in claim 1, it is characterized in that described phosphoric acid buffer is by sodium-chlor 6 ~ 10g, Repone K 0.05 ~ 0.5g, Sodium phosphate dibasic 1 ~ 1.2g, potassium primary phosphate 0.05 ~ 0.5g, calcium chloride 0.05 ~ 0.2g and being dissolved in 1000ml distilled water containing the magnesium chloride 0.05 ~ 0.2g of 6 crystal water obtains.
4. a kind of infections chicken cloacal bursa virus diluent as claimed in claim 1, is characterized in that the component containing following weight proportion in described every 1000ml phosphoric acid buffer: combination of amino acids 1.2 ~ 1.6g, VITAMIN combination 1.5 ~ 2.5g, Sodium Selenite 14 ~ 16mg and herbal mixture polysaccharide 14 ~ 16g.
5. a kind of infections chicken cloacal bursa virus diluent as claimed in claim 1, it is characterized in that described combination of amino acids contains the amino acid of following weight percent content: Gelucystine 20 ~ 50%, α-amino-isovaleric acid 20 ~ 40% and Isoleucine 30 ~ 60%, be preferably Gelucystine 20 ~ 35%, α-amino-isovaleric acid 25 ~ 35% and Isoleucine 40 ~ 50%.
6. a kind of infections chicken cloacal bursa virus diluent as claimed in claim 1, it is characterized in that the described VITAMIN of VITAMIN combination containing following weight percent content: VB11 5 ~ 40%, Catergen 0 ~ 50% and vitamin D2 0 ~ 40%, be preferably vitamin B12 0 ~ 35%, vitamins C 30 ~ 45% and Vitamin D3 500,000 I.U/GM 0 ~ 35%.
7. a kind of infections chicken cloacal bursa virus diluent as claimed in claim 1, is characterized in that described herbal mixture polysaccharide obtains according to the following steps:
A, take each Chinese medicine material by Flos Inulae 20 ~ 40 parts, White Mustard Seed 5 ~ 20 parts, Rhizoma Typhonii 10 ~ 30 parts, Herba Lycopi 5 ~ 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 liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out alcohol precipitation, precipitation obtains rough herbal polysaccharide;
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 gac 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, supernatant liquor in d carried out 10 times and concentrate, obtained herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out vacuum lyophilization obtain compound Chinese medicine polysaccharide frozen dried powder.
8. an infections chicken cloacal bursa virus diluent preparation method, is characterized in that comprising following processing step:
1) preparation of phosphate buffer solution
By sodium-chlor 6 ~ 10g, Repone K 0.05 ~ 0.5g, Sodium phosphate dibasic 1 ~ 1.2g, potassium primary phosphate 0.05 ~ 0.5g, calcium chloride 0.05 ~ 0.2g and be dissolved in 1000ml distilled water containing the magnesium chloride 0.05 ~ 0.2g of 6 crystal water, by 0.22 μm of filter membrane under aseptic condition, filtration sterilization is for subsequent use;
2) preparation of herbal mixture polysaccharide
A, take each raw material by Flos Inulae 20 ~ 40 parts, White Mustard Seed 5 ~ 20 parts, Rhizoma Typhonii 10 ~ 30 parts, Herba Lycopi 5 ~ 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 liquid, after room temperature cooling through 10000rpm centrifugal 10 minutes, discard precipitation, collect supernatant;
C, supernatant in b is carried out alcohol precipitation, precipitation obtains rough herbal polysaccharide;
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 gac 4 DEG C of attach overnight in 0.5% ratio, after absorption through 10000rpm centrifugal 10 minutes, discard precipitation, collection supernatant;
E, supernatant liquor in d carried out 10 times and concentrate, obtained herbal polysaccharide concentrated solution;
F, herbal polysaccharide concentrated solution in e is carried out vacuum lyophilization obtain compound Chinese medicine polysaccharide frozen dried powder;
3) inoculated into chick embryo viral dilution liquid is prepared: by combination of amino acids 1 ~ 2g, VITAMIN combination 1 ~ 3g, Sodium Selenite 10 ~ 20mg and herbal mixture polysaccharide 10 ~ 20g, be dissolved in the obtained phosphate buffer solution of step 1), abundant mixing, regulate between pH to 6.2 ~ 7.2 with acid or alkali, by 0.22 μm of filter membrane under aseptic condition, rearmounted 4 DEG C of filtration sterilization saves backup.
9. a kind of infections chicken cloacal bursa virus diluent preparation method as claimed in claim 8, it is characterized in that in described step 3), combination of amino acids contains the amino acid of following weight percent content: Gelucystine 20 ~ 50%, α-amino-isovaleric acid 20 ~ 40% and Isoleucine 30 ~ 60%, be preferably Gelucystine 20 ~ 35%, α-amino-isovaleric acid 25 ~ 35% and Isoleucine 40 ~ 50%.
10. a kind of infections chicken cloacal bursa virus diluent preparation method as claimed in claim 8, it is characterized in that the VITAMIN of VITAMIN combination containing following weight percent content in described step 3): VB11 5 ~ 40%, Catergen 0 ~ 50% and vitamin D2 0 ~ 40%, be preferably vitamin B12 0 ~ 35%, vitamins C 30 ~ 45% and Vitamin D3 500,000 I.U/GM 0 ~ 35%.
CN201511006412.7A 2015-12-29 2015-12-29 Infectious bursa disease virus diluent and preparation method thereof Pending CN105505888A (en)

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Citations (5)

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CN101690812A (en) * 2009-10-21 2010-04-07 辽宁益康生物制品有限公司 Concentrated freeze-dried yolk antibody composite preparation for Newcastle disease-infectious bursal disease and preparation process thereof
CN103409375A (en) * 2013-08-22 2013-11-27 浙江美保龙生物技术有限公司 Virus diluent for inoculating chick embryo and preparation method of virus diluent
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Application publication date: 20160420