CN103409375B - Virus diluent for inoculating chick embryo and preparation method of virus diluent - Google Patents
Virus diluent for inoculating chick embryo and preparation method of virus diluent Download PDFInfo
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- CN103409375B CN103409375B CN201310369029.2A CN201310369029A CN103409375B CN 103409375 B CN103409375 B CN 103409375B CN 201310369029 A CN201310369029 A CN 201310369029A CN 103409375 B CN103409375 B CN 103409375B
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Abstract
The invention discloses a virus diluent for inoculating a chick embryo and a preparation method of the virus diluent, belonging to the technical field of animal biological products. The virus diluent for inoculating the chick embryo contains the following components in weight ratio: 10-50g of glucose, 3-5g of amino acid composition, 6-10g of vitamin composition, 0-10mg of sodium selenite and 30-100g of compound traditional Chinese medicine polysaccharide in every 1000ml of phosphate buffer solution. After a virus diluted by using the virus diluent is inoculated to the chick embryo, the virus titer and the quantity of the obtained virus liquid can be simultaneously increased.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of inoculated into chick embryo viral dilution liquid and preparation method thereof.
Background technology
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 newcastle disease vaccine 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.
Glucose has critical role in field of biology, is the energy derive of viable cell and metabolic intermediate product.Be that one can directly absorb, supplementing the carbohydrate of heat energy, is the main source of body institute energy requirement.The function of detoxification of liver can be promoted, have provide protection to liver.Energy goods and materials the most common in organism.
Amino acid is the common name of the class organic compound containing amino and carboxyl.The basic composition unit of biological function macro-molecular protein is the base substance forming Animal nutrition desired protein.VITAMIN has another name called vitamin b6 usp, popular, the material namely sustained life, and is to maintain the necessary type organic matter of body vital movement, is also the important active substances keeping body health.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 katalysis 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 eubolism.Selenium and vitamin-E are worked in coordination with in vivo, can Cell protection film, prevent the oxidation of unsaturated fatty acids.
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 biological phenomena.Polysaccharose substance is the important component part of all Living organisms, has scavenging free radicals, improves the ability of activities of antioxidant enzymes and anti-lipid peroxidation.
The present invention is based on above-mentioned technical background, a kind of inoculated into chick embryo viral dilution liquid is proposed, specifically be specially adapted to a diluent for newcastle disease virus, solve a cultivation difficult problem for Avian pneumo-encephalitis virus low day instar chicken embryo, improve virus liquid harvest yield and virus titer.
Summary of the invention
For prior art Problems existing, the object of the invention is to design the technical scheme that a kind of inoculated into chick embryo viral dilution liquid and preparation method thereof is provided, by inoculated into chick embryo after this viral dilution liquid virus dilution, virus titer and the viral liquid measure of results can be improved simultaneously.
Described a kind of inoculated into chick embryo viral dilution liquid, is characterized in that the component containing following weight proportion in every 1000ml phosphoric acid buffer: glucose 10 ~ 50g, combination of amino acids 3 ~ 5g, VITAMIN combination 6 ~ 10g, Sodium Selenite 0 ~ 10mg and herbal mixture polysaccharide 30 ~ 100g;
Described combination of amino acids comprises L-PROLINE, glycine, tyrosine, Gelucystine and Serine;
Described VITAMIN combination comprises VITMAIN B1, vitamins C and vitamin-E;
Described herbal mixture polysaccharide is extracted by Radix Achyranthis Bidentatae, Chinese caterpillar fungus, Poria cocos, matrimony vine and the red sage root and obtains.
Described a kind of inoculated into chick embryo viral dilution liquid, is characterized in that the pH of this diluent is 6.6 ~ 7.
Described a kind of inoculated into chick embryo viral dilution liquid, 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 inoculated into chick embryo viral dilution liquid, is characterized in that the component containing following weight proportion in every 1000ml phosphoric acid buffer: glucose 15 ~ 45g, combination of amino acids 3.5 ~ 4.5g, VITAMIN combination 7 ~ 9g, Sodium Selenite 2 ~ 8mg and herbal mixture polysaccharide 40 ~ 80g.
Described a kind of inoculated into chick embryo viral dilution liquid, is characterized in that containing in described combination of amino acids: L-PROLINE 10 ~ 30%, glycine 5 ~ 15%, tyrosine 10 ~ 30%, Gelucystine 20 ~ 40% and Serine 10 ~ 30%.
Described a kind of inoculated into chick embryo viral dilution liquid, is characterized in that containing in described combination of amino acids: L-PROLINE 12 ~ 28%, glycine 8 ~ 12%, tyrosine 15 ~ 25%, Gelucystine 25 ~ 35% and Serine 15 ~ 25%.
Described a kind of inoculated into chick embryo viral dilution liquid, is characterized in that containing in described VITAMIN combination: VITMAIN B1 15 ~ 35%, vitamins C 40 ~ 70% and vitamin-E 10 ~ 30%.
Described a kind of inoculated into chick embryo viral dilution liquid, is characterized in that containing in described VITAMIN combination: VITMAIN B1 20 ~ 30%, vitamins C 50 ~ 60% and vitamin-E 10 ~ 20%.
Described a kind of inoculated into chick embryo viral dilution liquid, is characterized in that described herbal mixture polysaccharide obtains according to the following steps:
A, take each Chinese medicine material by Radix Achyranthis Bidentatae 20 ~ 40 parts, Chinese caterpillar fungus 10 ~ 30 parts, 10 ~ 30 parts, Poria cocos, matrimony vine 5 ~ 20 parts and the red sage root 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 inoculated into chick embryo viral dilution liquid, 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 Radix Achyranthis Bidentatae, Chinese caterpillar fungus, Poria cocos, matrimony vine and the red sage 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 glucose, 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 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 2 ~ 8 times.
(4) 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 20% ~ 30%.
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.The per-cent related in the present invention is all weight percentage.
Embodiment 1
A kind of inoculated into chick embryo viral dilution liquid, contain in the every 1000ml phosphoric acid buffer of this viral dilution liquid: glucose 25g, combination of amino acids 3.3g(wherein L-PROLINE 0.8g, glycine 0.2g, tyrosine 0.5g, Gelucystine 1.2g, Serine 0.6g), VITAMIN combination 7g(wherein VITMAIN B1 2g, vitamins C 4g, vitamin-E 1g), herbal mixture polysaccharide 50g, Sodium Selenite 3mg, this diluent pH is 6.8.
A preparation method for inoculated into chick embryo viral dilution liquid, 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 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, 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 of weight 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 glucose 25g, L-PROLINE 0.8g, glycine 0.2g, tyrosine 0.5g, Gelucystine 1.2g, Serine 0.6g, VITMAIN B1 2g, vitamins C 4g, vitamin-E 1g, herbal mixture polysaccharide 50g by above-mentioned preferred diluent number of components, Sodium Selenite 3mg, be dissolved in the 1000ml phosphate buffer solution in step (1) in proportion, abundant mixing, pH to 6.8 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 inoculated into chick embryo viral dilution liquid, contain in the every 1000ml phosphoric acid buffer of this viral dilution liquid: glucose 40g, combination of amino acids 4g(wherein L-PROLINE 1g, glycine 0.4g, tyrosine 0.5g, Gelucystine 1.4g, Serine 0.7g), VITAMIN combination 8g(wherein VITMAIN B1 2g, vitamins C 4.5g, vitamin-E 1.5g), herbal mixture polysaccharide 60g, Sodium Selenite 8mg, this diluent pH is 7.0.
A preparation method for inoculated into chick embryo viral dilution liquid, its step is with embodiment 1.
Embodiment 3
A kind of inoculated into chick embryo viral dilution liquid, contain in the every 1000ml phosphoric acid buffer of this viral dilution liquid: glucose 50g, combination of amino acids 5g(wherein L-PROLINE 1.5g, glycine 0.75g, tyrosine 0.5g, Gelucystine 1g, Serine 1.25g), VITAMIN combination 10g(wherein VITMAIN B1 3.5g, vitamins C 5.5g, vitamin-E 1g), herbal mixture polysaccharide 100g, Sodium Selenite 10mg, this diluent pH is 6.6.Above-mentioned phosphoric acid buffer contains sodium-chlor 6g, Repone K 0.05g, Sodium phosphate dibasic 1g, potassium primary phosphate 0.05g, calcium chloride 0.05g, containing the magnesium chloride 0.05g of 6 crystal water.
A preparation method for inoculated into chick embryo viral dilution liquid, its step is with embodiment 1.
Embodiment 4
A kind of inoculated into chick embryo viral dilution liquid, contain in the every 1000ml phosphoric acid buffer of this viral dilution liquid: glucose 10g, combination of amino acids 3g(wherein L-PROLINE 0.3g, glycine 0.15g, tyrosine 0.9g, Gelucystine 1.2g, Serine 0.45g), VITAMIN combination 6g(wherein VITMAIN B1 0.9g, vitamins C 4.2g, vitamin-E 0.9g), herbal mixture polysaccharide 30g, Sodium Selenite 2mg, this diluent pH is 6.8.Above-mentioned every 1000ml phosphoric acid buffer contains sodium-chlor 10g, Repone K 0.5g, Sodium phosphate dibasic 1.2g, potassium primary phosphate 0.5g, calcium chloride 0.2g, containing the magnesium chloride 0.2g of 6 crystal water.
A preparation method for inoculated into chick embryo viral dilution liquid, its step is with embodiment 1.
Embodiment 5: a kind of using method of inoculated into chick embryo viral dilution liquid
1) inoculated into chick embryo viral dilution liquid and preparation thereof, method is with embodiment 1.
2) egg inoculation of Avian pneumo-encephalitis 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 (as 10 with above-mentioned viral dilution liquid
-4or 10
-5), inoculation 0.2ml 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 before 60 hours is discarded.After 60 hours, once, dead chicken embryo takes out every 4 ~ 8 hours photograph eggs at any time, until 120 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 by toxic chicken blastochyle amount sterile saline, get 10
-7, 10
-8, 10
-9inoculation 10 age in days SPF chicken embryo 5 in 3 each allantoic cavities of extent of dilution, every embryo 0.1ml, puts 36 ~ 37 DEG C and continues to hatch.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 answers>=10
8eID
50.
Comparative example 1: adopt comparing of above-mentioned diluted virus inoculation chicken embryo and conventional diluted virus inoculation chicken embryo indices.
1) inoculated into chick embryo viral dilution liquid of the present invention and preparation thereof, method is with embodiment 1.
2) conventional viral dilution liquid and preparation, i.e. physiological saline, technology is prepared routinely.
3) egg inoculation of Avian pneumo-encephalitis virus is bred with cultivation
(1) selection of inoculated into chick embryo: select well-developed 9 age in days SPF chicken embryos, 2000 pieces; Select well-developed 10 age in days SPF chicken embryos, 1000 pieces.
(2) inoculate: 2000 piece of 9 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 viral dilution and inoculation, second group adopts the viral dilution liquid of routine techniques preparation to carry out viral dilution and inoculation, two groups of extension rates are identical, and inoculation method is with embodiment 5; Using 1000 piece of 10 age in days SPF chicken embryo in this example (1) as the 3rd group, adopt the viral dilution liquid of routine techniques preparation to carry out viral dilution and inoculation, extension rate is identical with the one the second groups, and inoculation method is with embodiment 5.
(3) hatch and observe, gather in the crops, viral suspension inspection, its method is all with embodiment 5.
4) dead time of concentration and quantity after three prescription method inoculated into chick embryo, in table 1.
First group: be diluent group of the present invention, adopt 9 age in days egg inoculations.
Second group: be routine techniques diluent group, adopt 9 age in days egg inoculations.
3rd group: be routine techniques diluent group, adopt 10 age in days egg inoculations.
5) virus harvest amount and viral level thereof after three prescription method inoculated into chick embryo, in table 2.
| Group | Results chicken embryo number (piece) | Total yield (ml) | Every piece of chicken embryo harvest yield (piece) | HA tires (2log) | EID 50/0.1ml |
| First group | 984 | 13700 | 13.9 | 13 | 10 9.6 |
| Second group | 955 | 8200 | 8.6 | 9 | 10 7.5 |
| 3rd group | 963 | 8700 | 9.0 | 10 | 10 8.4 |
In sum, compared with conventional art, diluent of the present invention is adopted to carry out viral dilution inoculation instar chicken embryo of low day, the chicken embryo survival time can be extended, extend the virus multiplication time, improve viral level 2 ~ 8 times, also improve chicken blastochyle harvest yield 20% ~ 30% simultaneously, this comparative example is also surprised to find that, adopts chicken embryo death quantity in diluted virus inoculation chicken embryo of the present invention 60 hours to decline to some extent, may be relevant with having antibacterial ability containing herbal polysaccharide in this diluent.
The diluent obtained in embodiment 2-4 compares the identical test of example 1, and its net result also can reach the technique effect identical with comparative example 1.
Claims (5)
1. an inoculated into chick embryo viral dilution liquid, it is characterized in that the component containing following weight proportion in every 1000ml phosphoric acid buffer: glucose 10 ~ 50g, combination of amino acids 3 ~ 5g, VITAMIN combination 6 ~ 10g, Sodium Selenite 0 ~ 10mg and herbal mixture polysaccharide 30 ~ 100g, the pH of this diluent is 6.6 ~ 7.0;
Contain in described combination of amino acids: L-PROLINE 10 ~ 30%, glycine 5 ~ 15%, tyrosine 10 ~ 30%, Gelucystine 20 ~ 40% and Serine 10 ~ 30%;
Contain in described VITAMIN combination: VITMAIN B1 15 ~ 35%, vitamins C 40 ~ 70% and vitamin-E 10 ~ 30%;
Described phosphoric acid buffer 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 herbal mixture polysaccharide obtains according to the following steps:
A, take each Chinese medicine material by Radix Achyranthis Bidentatae 20 ~ 40 parts, Chinese caterpillar fungus 10 ~ 30 parts, 10 ~ 30 parts, Poria cocos, matrimony vine 5 ~ 20 parts and the red sage root 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.
2. a kind of inoculated into chick embryo viral dilution liquid as claimed in claim 1, is characterized in that the component containing following weight proportion in every 1000ml phosphoric acid buffer: glucose 15 ~ 45g, combination of amino acids 3.5 ~ 4.5g, VITAMIN combination 7 ~ 9g, Sodium Selenite 2 ~ 8mg and herbal mixture polysaccharide 40 ~ 80g.
3. a kind of inoculated into chick embryo viral dilution liquid as claimed in claim 1, is characterized in that containing in described combination of amino acids: L-PROLINE 12 ~ 28%, glycine 8 ~ 12%, tyrosine 15 ~ 25%, Gelucystine 25 ~ 35% and Serine 15 ~ 25%.
4. a kind of inoculated into chick embryo viral dilution liquid as claimed in claim 1, is characterized in that containing in described VITAMIN combination: VITMAIN B1 20 ~ 30%, vitamins C 50 ~ 60% and vitamin-E 10 ~ 20%.
5. the preparation method of a kind of inoculated into chick embryo viral dilution liquid 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 Radix Achyranthis Bidentatae, Chinese caterpillar fungus, Poria cocos, matrimony vine and the red sage 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 glucose, 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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| CN105441394A (en) * | 2015-12-29 | 2016-03-30 | 浙江美保龙生物技术有限公司 | Avian influenza virus diluent and preparation method thereof |
| CN105497889A (en) * | 2015-12-29 | 2016-04-20 | 浙江美保龙生物技术有限公司 | Preparing method for newcastle disease and bird flu bigeminy compound inactivated vaccine |
| CN111057683A (en) * | 2019-12-25 | 2020-04-24 | 乾元浩生物股份有限公司 | Virus diluent for chick embryo inoculation and preparation method and application thereof |
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