CN105368790A - Chicken infectious bronchitis virus diluent and preparation method thereof - Google Patents
Chicken infectious bronchitis virus diluent and preparation method thereof Download PDFInfo
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- CN105368790A CN105368790A CN201510692414.XA CN201510692414A CN105368790A CN 105368790 A CN105368790 A CN 105368790A CN 201510692414 A CN201510692414 A CN 201510692414A CN 105368790 A CN105368790 A CN 105368790A
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Abstract
The invention relates to a chicken infectious bronchitis virus diluent and a preparation method thereof and belongs to the technical field of animal biological products. Every 1000 ml of phosphate buffer in the diluent comprises, by weight, 1-4 g of amino acid combination, 1-5 g of vitamin combination, 12-18 mg of sodium selenite and 5-25 g of compound traditional Chinese medicine polysaccharide. The amino acid combination comprises methionine, tryptophan, tyrosine and leucine. The vitamin combination comprises vitamin B2, vitamin D and vitamin E. The compound traditional Chinese medicine polysaccharide is obtained by extraction from curculigo orchioides, eucommia ulmoides, poria cocos and dipsacus asperoids. After chick embryos are inoculated with the diluent, the virus resistance of the chick embryos can be improved, the death time is prolonged, and the virus multiplication time is prolonged. Compared with the prior art, the virus titer is improved by twice to eight times. According to the prepared diluent, the low-day-old chick embryos are inoculated, it is incidentally found that the size of obtained allantoic fluid is greatly increased, and the size of the obtained allantoic fluid is increased by 30-40%.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of avian infectious bronchitis virus diluent and preparation method thereof.
Background technology
Chicken infectious bronchitis (IB), it is the virus disease that a kind of acute, the high degree in contact of the chicken caused by infectious bronchitis virus (IBV) infect, with tracheal rale, coughing, sneeze for principal character, is one of main epidemic disease of harm poultry husbandry.The serotype of chicken infectious bronchitis is numerous, the serotype reported at present has more than 30 to plant, different serotypes strain also exists very big-difference in virulence, pathogenic and tissue tropism, do not have each other or only have partial intersection immunity, constantly occur with stylish serotype, bring many difficulties to the prevention of IBV.
The proviral cultivation of order has animal inoculation pvaccination, tissue culture and chick embryo culture 3 kinds of methods.Wherein chick embryo culture is one of method the most frequently used in virus culture.Chicken embryo is just at developmental body, and many animals virus can be bred and go down to posterity in chicken embryo.The advantage of chicken embryo is that the degree of tissue differentiation of embryo is low, and different ages in days and route of inoculation can be selected, virus is easy to propagation in chicken embryo, and fractionated viral infected chicken embryo can produce bean cotyledon later, causes the specific infection indications such as hyperemia, hemorrhage, necrosis region and death.Infect containing a large amount of virus in the chicken embryo tissue of virus and liquid, easy acquisition and processing, and source is sufficient, equipment and easy to operation.
The chicken infectious bronchitis that Present Domestic is produced is substantially all breed after adopting egg inoculation virus, and results allantoic fluid is prepared from.Research shows, chicken embryo age in days and chick embryo allantois liquid measure when planting malicious inoculum size and results are negative correlation.Namely along with inoculated into chick embryo age in days increase and plant the raising of malicious inoculum size, the chick embryo allantoic liquid of results can reduce.Usually instar chicken embryo on the 10th is selected to carry out the breeding of Avian pneumo-encephalitis virus inoculation culture, generally in cultivation 96 ~ 120 hours results chicken blastochyles, i.e. 14 ~ 15 ages in days, now age in days is bigger than normal, allantoic fluid content not high (13 ~ 14 age in days allantoic fluid content of chick embryo development are the highest); If improve allantoic fluid content must gather in the crops in advance, but now viral level does not reach peak value, causing malicious valency to reduce loses more than gain equally.The degree of tissue differentiation of age in days lower chicken embryo is lower in addition, more be conducive to the propagation of virus, but adopt low age in days (as 9 ages in days) chicken embryo to carry out the inoculation culture of virus by usual means, because chicken embryo resistibility is poor, premature death etc. cause viral titer in chick embryo allantoic liquid lower, and allantoic fluid content is also lower.
Summary of the invention
For prior art Problems existing, the object of the invention is to design the technical scheme that a kind of avian infectious bronchitis virus diluent and preparation method thereof is provided.
Described a kind of avian infectious bronchitis virus diluent, it is characterized in that the component containing following weight proportion in every 1000ml phosphoric acid buffer: combination of amino acids 1 ~ 4g, VITAMIN combination 1 ~ 5g, Sodium Selenite 12 ~ 18mg and herbal mixture polysaccharide 5 ~ 25g, described combination of amino acids comprises methionine(Met), tryptophane, tyrosine and leucine, described VITAMIN combination comprises Lin Suanna Vitamin B2 Sodium Phosphate, vitamins D and vitamin-E, and described herbal mixture polysaccharide is extracted by thizoma curculiginis, the bark of eucommia, Poria cocos and teasel root and obtains.
Described a kind of avian infectious bronchitis virus diluent, is characterized in that the pH of diluent is 6.6 ~ 7.
Described a kind of avian infectious bronchitis 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, the magnesium chloride 0.05 ~ 0.2g containing 6 crystal water is dissolved in 1000ml distilled water and obtains.
Described a kind of avian infectious bronchitis virus diluent, is characterized in that the component containing following weight proportion in described every 1000ml phosphoric acid buffer: combination of amino acids 2 ~ 3g, VITAMIN combination 2 ~ 4g, Sodium Selenite 14 ~ 16mg and herbal mixture polysaccharide 10 ~ 20g.
Described a kind of avian infectious bronchitis virus diluent, it is characterized in that described combination of amino acids comprises the amino acid of following weight percent: methionine(Met) 20 ~ 30%, tryptophane 20 ~ 40%, tyrosine 10 ~ 30%, leucine 10 ~ 30%, be preferably methionine(Met) 25 ~ 30%, tryptophane 25 ~ 35%, tyrosine 15 ~ 25%, leucine 15 ~ 25%.
Described a kind of avian infectious bronchitis virus diluent, it is characterized in that described VITAMIN combination comprises the VITAMIN of following weight percent: Lin Suanna Vitamin B2 Sodium Phosphate 15 ~ 40%, vitamin D2 0 ~ 50% and vitamin E2 0 ~ 40%, be preferably Lin Suanna Vitamin B2 Sodium Phosphate 20 ~ 35%, Vitamin D3 500,000 I.U/GM 0 ~ 45% and vitamin-E 30 ~ 35%.
Described a kind of avian infectious bronchitis virus diluent, is characterized in that described herbal mixture polysaccharide obtains according to the following steps:
A, take each Chinese medicine material by thizoma curculiginis 10 ~ 30 parts, the bark of eucommia 10 ~ 20 parts, 20 ~ 40 parts, Poria cocos and interrupted 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.
The preparation method of described a kind of avian infectious bronchitis virus diluent, is characterized in that comprising following processing step:
1) preparation of phosphate buffer solution: take sodium-chlor, Repone K, Sodium phosphate dibasic, potassium primary phosphate, calcium chloride in proportion, be dissolved in distilled water containing the magnesium chloride 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 thizoma curculiginis, the bark of eucommia, Poria cocos and teasel root in proportion, chopping, clean after spend the night by cold water soak, then add the purified water of raw material weight 15 times, 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 preparation: take combination of amino acids, VITAMIN combination, herbal mixture polysaccharide and Sodium Selenite in proportion, be dissolved in the obtained phosphate buffer solution of step 1), abundant mixing, regulate between pH to 6.6 ~ 7.0 with acid or alkali, by 0.22 μm of filter membrane under aseptic condition, rearmounted 4 DEG C of filtration sterilization saves backup.
The present invention has actively useful effect:
(1) 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.
(2), 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 2 ~ 8 times.
(3) diluent prepared by the present invention, inoculate low day instar chicken embryo, find that the allantoic fluid volume gathered in the crops significantly increases unexpectedly, compared with prior art, the allantoic fluid volume obtained improves 30 ~ 40%.
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 avian infectious bronchitis virus diluent, contain in the every 1000ml phosphoric acid buffer of this viral dilution liquid: combination of amino acids 3g(is methionine(Met) 0.75g, tryptophane 0.9g, tyrosine 0.75g, Gelucystine 0.6g wherein), VITAMIN combination 4g(wherein Lin Suanna Vitamin B2 Sodium Phosphate 1.2g, Dry Vitamin D3 100 cws .6g and vitamin e1 .2g), herbal mixture polysaccharide 20g, Sodium Selenite 16mg, this diluent pH is 7.0.
A preparation method for avian infectious bronchitis 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 thizoma curculiginis 20 parts, the bark of eucommia 15 parts, 30 parts, Poria cocos and interrupted 15 parts, chopping, clean after spend the night by cold water soak, then add the purified water of raw material weight 15 times, 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 each composition, be dissolved in proportion in the 1000ml phosphate buffer solution in step (1), fully mix, regulate pH to 7.0 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 avian infectious bronchitis 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 4g(is methionine(Met) 0.8g wherein, tryptophane 0.8g, tyrosine 1.2g, Gelucystine 1.2g), VITAMIN combination 5g(wherein Lin Suanna Vitamin B2 Sodium Phosphate 2g, Dry Vitamin D3 100 cws g and vitamin E2 g), herbal mixture polysaccharide 25g(is from thizoma curculiginis 10 parts, the bark of eucommia 10 parts, extract in 20 parts, Poria cocos and interrupted 5 parts and obtain), Sodium Selenite 12mg, this diluent pH is 7.0.
A preparation method for avian infectious bronchitis virus diluent, its step is with embodiment 1.
Embodiment 3
A kind of avian infectious bronchitis 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 1g(is methionine(Met) 0.3g wherein, tryptophane 0.4g, tyrosine 0.1g, Gelucystine 0.2g), VITAMIN combination 1g(wherein Lin Suanna Vitamin B2 Sodium Phosphate 0.15g, vitamins D 0.5g and vitamin-E 0.35g), herbal mixture polysaccharide 5g(is from thizoma curculiginis 30 parts, the bark of eucommia 20 parts, extract in 40 parts, Poria cocos and interrupted 20 parts and obtain), Sodium Selenite 18mg, this diluent pH is 6.6.
A preparation method for avian infectious bronchitis virus diluent, its step is with embodiment 1.
Embodiment 4: the using method of avian infectious bronchitis virus diluent
1) avian infectious bronchitis virus diluent preparing, method is with embodiment 1.
2) egg inoculation of avian infectious bronchitis 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, do suitably dilution with diluent, inoculation 0.1ml in every embryo allantoic cavity.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 is before 48h discarded.After 48 hours, once, dead chicken embryo takes out every 4 ~ 8 hours photograph eggs at any time, until 96 hours, no matter death whether, is all taken out, air chamber is upwards upright, is placed in 2 ~ 8 DEG C of coolings.
(4) gather in the crops
The cooling chicken embryo of 4 ~ 24 hours is taken out, with iodine tincture disinfection air chamber position, then divests air chamber portion chorion with aseptic operation, throw off shell membrane, break chorioallantoic membrane and amnion (not making yolk break), draw chicken blastochyle, often the blastochyle of several chicken embryos is mixed into one group.Blastochyle after results is placed in sterilising vessel, adds suitable microbiotic, 2 ~ 8 DEG C of icebox process.After keeping sample, preservation of freezing.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 suspension inspection
Steriling test: processed blastochyle, often organizes and samples respectively, carries out steriling test by " steriling test or pure checked operation code ", should without bacterial growth.
Viral level measures: make 10 times of serial dilutions to 10 by toxic chicken blastochyle amount sterile saline
-4, get inoculation 10 age in days SPF chicken embryo 5 in 3 each allantoic cavities of acceptable diluent degree, every embryo 0.1ml, puts 36 ~ 37 DEG C and continues to hatch 6.Chicken embryo dead before 48 hours discards to be disregarded, the chicken embryo dead at 48 ~ 120 hours takes out at any time, results chicken blastochyle, same dilution blastochyle balanced mix, measures red cell agglutination valency respectively by extent of dilution, to 120 hours, take out all embryos alive, gather in the crops chicken blastochyle one by one, measure red cell agglutination valency respectively, agglutination titer>=1:160(micromethod 1:128) person is judged to infection, calculates EID
50, every 0.1ml viral level should be not less than 10
8eID
50.
Comparative example 1: adopt comparing of diluted virus inoculation chicken embryo of the present invention and conventional diluted virus inoculation chicken embryo indices.
1) the avian infectious bronchitis 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 is divided into 2 groups, first group adopts viral dilution liquid of the present invention to carry out operation inoculation, and 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.
Comparative example 2: adopt stability between diluted virus inoculation chicken embryo of the present invention and conventional diluted virus inoculation chicken embryo batch to compare.
Carry out the cultivation in the same enbryo experiment of three batches of infectious bronchitis virus continuously by comparative example 1 method, experimental result is in table 3.
In sum, compared with conventional art, adopt the inventive method can improve viral level 2 ~ 8 times, also improve chicken blastochyle harvest yield 20% ~ 30% simultaneously.
Embodiment 2 and the diluent obtained in 3 compare example 1, test that comparative example 2 is identical, and its net result also can reach the technique effect identical with comparative example 1, can improve viral level more than 8 times, also improves chicken blastochyle harvest yield 30% ~ 40% simultaneously.
Claims (8)
1. an avian infectious bronchitis virus diluent, it is characterized in that the component containing following weight proportion in every 1000ml phosphoric acid buffer: combination of amino acids 1 ~ 4g, VITAMIN combination 1 ~ 5g, Sodium Selenite 12 ~ 18mg and herbal mixture polysaccharide 5 ~ 25g, described combination of amino acids comprises methionine(Met), tryptophane, tyrosine and leucine, described VITAMIN combination comprises Lin Suanna Vitamin B2 Sodium Phosphate, vitamins D and vitamin-E, and described herbal mixture polysaccharide is extracted by thizoma curculiginis, the bark of eucommia, Poria cocos and teasel root and obtains.
2. a kind of avian infectious bronchitis virus diluent as claimed in claim 1, is characterized in that the pH of diluent is 6.6 ~ 7.
3. a kind of avian infectious bronchitis 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, the magnesium chloride 0.05 ~ 0.2g containing 6 crystal water is dissolved in 1000ml distilled water and obtains.
4. a kind of avian infectious bronchitis 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 2 ~ 3g, VITAMIN combination 2 ~ 4g, Sodium Selenite 14 ~ 16mg and herbal mixture polysaccharide 10 ~ 20g.
5. a kind of avian infectious bronchitis virus diluent as claimed in claim 1, it is characterized in that described combination of amino acids comprises the amino acid of following weight percent: methionine(Met) 20 ~ 30%, tryptophane 20 ~ 40%, tyrosine 10 ~ 30%, leucine 10 ~ 30%, be preferably methionine(Met) 25 ~ 30%, tryptophane 25 ~ 35%, tyrosine 15 ~ 25%, leucine 15 ~ 25%.
6. a kind of avian infectious bronchitis virus diluent as claimed in claim 1, it is characterized in that described VITAMIN combination comprises the VITAMIN of following weight percent: Lin Suanna Vitamin B2 Sodium Phosphate 15 ~ 40%, vitamin D2 0 ~ 50% and vitamin E2 0 ~ 40%, be preferably Lin Suanna Vitamin B2 Sodium Phosphate 20 ~ 35%, Vitamin D3 500,000 I.U/GM 0 ~ 45% and vitamin-E 30 ~ 35%.
7. a kind of avian infectious bronchitis 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 thizoma curculiginis 10 ~ 30 parts, the bark of eucommia 10 ~ 20 parts, 20 ~ 40 parts, Poria cocos and interrupted 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. the preparation method of a kind of avian infectious bronchitis virus diluent as claimed in claim 1, is characterized in that comprising following processing step:
1) preparation of phosphate buffer solution: take sodium-chlor, Repone K, Sodium phosphate dibasic, potassium primary phosphate, calcium chloride in proportion, be dissolved in distilled water containing the magnesium chloride 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 thizoma curculiginis, the bark of eucommia, Poria cocos and teasel root in proportion, chopping, clean after spend the night by cold water soak, then add the purified water of raw material weight 15 times, 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 preparation: take combination of amino acids, VITAMIN combination, herbal mixture polysaccharide and Sodium Selenite in proportion, be dissolved in the obtained phosphate buffer solution of step 1), abundant mixing, regulate between pH to 6.6 ~ 7.0 with acid or alkali, by 0.22 μm of filter membrane under aseptic condition, rearmounted 4 DEG C of filtration sterilization saves backup.
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| CN101491674A (en) * | 2008-12-26 | 2009-07-29 | 天津瑞普生物技术股份有限公司 | Production technique of dual vaccine of chicken new castle disease and infectious bronchitis |
| 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 |
| CN103599533A (en) * | 2013-09-23 | 2014-02-26 | 天津瑞普生物技术股份有限公司 | Chicken new castle disease-infectious bronchitis trivalent combined vaccine and preparation method thereof |
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Application publication date: 20160302 |