CN104560892A - Culture method for increasing virus titer of duck flavivirus - Google Patents
Culture method for increasing virus titer of duck flavivirus Download PDFInfo
- Publication number
- CN104560892A CN104560892A CN201510050831.4A CN201510050831A CN104560892A CN 104560892 A CN104560892 A CN 104560892A CN 201510050831 A CN201510050831 A CN 201510050831A CN 104560892 A CN104560892 A CN 104560892A
- Authority
- CN
- China
- Prior art keywords
- affinity agent
- nutrient solution
- cultural method
- cell
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a culture method for increasing the virus titer of duck flavivirus, and belongs to the field of microbial virus technologies. The culture method mainly comprises the following steps: preparing a DMEM culture solution; after fetal calf serums are added, inoculating cells for carrying out monolayer culture; after an affinity absorber and the DMEM culture solution are added, further culturing for 1-30 min, and inoculating duck flavivirus for 2-5 days, so that duck flavivirus liquid with a high virus titer can be obtained. The culture method disclosed by the invention increases the contact affinity of viruses and cells mainly by changing a microenvironment between the duck flavivirus and the cells, thereby greatly increasing the virus titer of the duck flavivirus.
Description
Technical field
The present invention relates to microbial virus technical field, be specifically related to a kind of cultural method improving duck flavivirus virus titre.
Background technology
Duck flavivirus (duck tembusu virus) a kind ofly egg duck can be caused to lay eggs the virus of function degradation, its clinical manifestation is that laying rate declines suddenly, egg-laying peak shortens, serious laying rate at short notice can reduce to zero, food consumption also together with time decline, have the phenomena of mortality individually.Pathology analyses the serious change such as the hyperemia of visible ovary, swelling, necrosis and atrophy.After this disease was found first at home since 2010, the egg duck cultivation large province egg ducks such as Shandong, Hunan, Guangdong have been caused to drop in production over a large area.At present, many research institutions have been separated to duck flavivirus clinical, and have carried out cell infection test in vitro, attempt to make this virus multiplication to the development for duck flavivirus vaccine by vitro culture.Many sections of bibliographical informations are pointed out, the clinical duck flavivirus be separated to can carry out Infection in Vitro to various kinds of cell, and as VERO cell, bhk cell etc., but primary viral culturing process is comparatively slow, virus titer is not high enough, and the research and development for vaccine are the problems needing to solve.
Summary of the invention
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a kind of cultural method improving duck flavivirus virus titre, cultural method of the present invention is mainly through changing duck flavivirus and intercellular microenvironment, increase virus and cells contacting affinity, thus greatly improve duck flavivirus virus titre.
For solving the problem, the technical solution adopted in the present invention is as follows:
Improve a cultural method for duck flavivirus virus titre, it comprises the following steps:
The preparation of A, DMEM nutrient solution: be dissolved in 1000ml deionized water by 13.4g DMEM dry powder, is stirred to after dissolving completely, adjust ph to 6.8 ~ 7.8, and carries out Sterile Filtration with the sterilised membrane filter of≤0.22um, be placed in≤refrigerator of 4 DEG C is stand-by;
B, in above-mentioned DMEM solution, add the foetal calf serum accounting for DMEM nutrient solution volume 1% ~ 8% after, inoculating cell carries out monolayer culture;
C, after monolayer cell culture is good, discards the DMEM nutrient solution in culture vessel, and wash 2 ~ 3 times by aseptic washing lotion;
D, discard aseptic washing lotion, in culture vessel, then add the DMEM nutrient solution described in affinity agent and steps A, wherein the volumetric usage of affinity agent is 0.01% ~ 1% of DMEM nutrient solution volumetric usage;
E, culture vessel is placed in cell culture incubator quiescent culture 1min ~ 30min after, inoculation duck flavivirus, mix be placed in cell culture incubator cultivate 2 ~ 5 days, results virus liquid.
Particularly, the cell described in step B is the one in Vero cell, 293 cells, ST cell, bhk cell.
Particularly, the aseptic washing lotion described in step C is the one in deionized water, PBS damping fluid, serum-free DMEM nutrient solution.
Particularly, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0 ~ 1%; Phosphoric acid 0 ~ 1%;
Sorbic Acid 0 ~ 0.5%; Oxysuccinic acid 0.01 ~ 1%;
Inorganic salt 0 ~ 3.5%; Deionized water surplus.
Preferably, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0.03 ~ 0.08%; Phosphoric acid 0.06 ~ 0.08%;
Sorbic Acid 0.1 ~ 0.3%; Oxysuccinic acid 0.03 ~ 0.08%;
Inorganic salt 0.05 ~ 3%; Deionized water surplus.
Preferably, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0.05%; Phosphoric acid 0.08%;
Sorbic Acid 0.3%; Oxysuccinic acid 0.05%;
Inorganic salt 0.2%; Deionized water 99.32%.
Particularly, described inorganic salt are one or more in sodium sulfate, Sodium phosphate dibasic, Repone K, sodium-chlor.
Particularly, described sodium sulfate accounts for 0.15 ~ 0.3% of affinity agent total amount; Described SODIUM PHOSPHATE, MONOBASIC accounts for the 0.1-1.6% of affinity agent total amount; Described sodium-chlor accounts for the 0.09-0.9% of affinity agent total amount; Described Repone K accounts for the 0.01-1% of affinity agent total amount.
Particularly, described affinity agent is prepared from according to the following steps: take propanedioic acid, phosphoric acid, Sorbic Acid, oxysuccinic acid, inorganic salt by formula ratio, said components is dissolved in the deionized water of formula ratio, filters and get final product.
Compared to existing technology, beneficial effect of the present invention is:
Cultural method of the present invention mainly passes through to add affinity agent in culturing process, change duck flavivirus and intercellular microenvironment, increase virus and iuntercellular affinity, duck flavivirus is made to infect Cells for production more rapidly, improve the efficiency of infection of duck flavivirus to cell, thus the duck flavivirus of high virus titer can be obtained in vitro in culture experiment more rapidly, for the vaccine research and development of duck flavivirus lay the foundation.
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment
Improve a cultural method for duck flavivirus virus titre, it comprises the following steps:
The preparation of A, DMEM nutrient solution: DMEM dry powder is dissolved in deionized water, be stirred to after dissolving completely, adjust ph to 6.8 ~ 7.8, and carry out Sterile Filtration with the sterilised membrane filter of 0.22um, the refrigerator being placed in 4 DEG C is stand-by;
B, in above-mentioned DMEM solution, add the foetal calf serum accounting for DMEM nutrient solution volume 1% ~ 8% after, inoculating cell carries out monolayer culture;
C, after monolayer cell culture is good, discards the DMEM nutrient solution in culture vessel, and wash 2 ~ 3 times by aseptic washing lotion;
D, discard aseptic washing lotion, in culture vessel, then add the DMEM nutrient solution described in affinity agent and steps A, wherein the volumetric usage of affinity agent is 0.01% ~ 1% of DMEM nutrient solution volumetric usage;
E, culture vessel is placed in cell culture incubator quiescent culture 1min ~ 30min after, inoculation duck flavivirus, mix be placed in cell culture incubator cultivate 2 ~ 5 days, results virus liquid.
Particularly, the cell described in step B is the one in Vero cell, 293 cells, ST cell, bhk cell.
Particularly, the aseptic washing lotion described in step C is the one in deionized water, PBS damping fluid, serum-free DMEM nutrient solution.
Particularly, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0 ~ 1%; Phosphoric acid 0 ~ 1%;
Sorbic Acid 0 ~ 0.5%; Oxysuccinic acid 0.01 ~ 1%;
Inorganic salt 0 ~ 3.5%; Deionized water surplus.
Preferably, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0.03 ~ 0.08%; Phosphoric acid 0.06 ~ 0.08%;
Sorbic Acid 0.1 ~ 0.3%; Oxysuccinic acid 0.03 ~ 0.08%;
Inorganic salt 0.05 ~ 3%; Deionized water surplus.
Preferably, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0.05%; Phosphoric acid 0.08%;
Sorbic Acid 0.3%; Oxysuccinic acid 0.05%;
Inorganic salt 0.2%; Deionized water 99.32%.
Particularly, described inorganic salt are one or more in sodium sulfate, Sodium phosphate dibasic, Repone K, sodium-chlor.
Particularly, described sodium sulfate accounts for 0.15 ~ 0.3% of affinity agent total amount; Described SODIUM PHOSPHATE, MONOBASIC accounts for the 0.1-1.6% of affinity agent total amount; Described sodium-chlor accounts for the 0.09-0.9% of affinity agent total amount; Described Repone K accounts for the 0.01-1% of affinity agent total amount.
Particularly, described affinity agent is prepared from according to the following steps: take propanedioic acid, phosphoric acid, Sorbic Acid, oxysuccinic acid, inorganic salt by formula ratio, said components is dissolved in the deionized water of formula ratio, filters and get final product.
Embodiment 1:
Improve a cultural method for duck flavivirus virus titre, it comprises the following steps:
The preparation of A, DMEM nutrient solution: 13.4g DMEM dry powder is dissolved in 1000ml deionized water, be stirred to after dissolving completely, adjust ph to 7.0, and carry out Sterile Filtration with 0.22um sterilised membrane filter, the refrigerator being placed in 4 DEG C is stand-by;
B, in above-mentioned DMEM solution, add the foetal calf serum accounting for DMEM nutrient solution volume 5% after, inoculation Vero cell carries out monolayer culture;
C, after Vero cell monolayer is cultivated, discard the DMEM nutrient solution in culture vessel, and wash 3 times with aseptic deionized water;
D, discard aseptic deionized water, in culture vessel, then add the DMEM nutrient solution described in affinity agent and steps A, wherein the volumetric usage of affinity agent is 1% of DMEM nutrient solution volumetric usage;
E, culture vessel is placed in cell culture incubator quiescent culture 30min after, inoculation duck flavivirus, mix be placed in cell culture incubator cultivate 3 days, results virus liquid.
Particularly, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0.5%; Phosphoric acid 0.5%;
Sorbic Acid 0.5%; Oxysuccinic acid 0.5%;
Inorganic salt 2.9%; Deionized water 95.5%.
Wherein, described inorganic salt are the mixing of sodium sulfate, Sodium phosphate dibasic, Repone K.
Wherein, described sodium sulfate accounts for 0.3% of affinity agent total amount; Described SODIUM PHOSPHATE, MONOBASIC accounts for 1.6% of affinity agent total amount; Described Repone K accounts for 1% of affinity agent total amount.
Particularly, described affinity agent is prepared from according to the following steps: take propanedioic acid, phosphoric acid, Sorbic Acid, oxysuccinic acid, sodium sulfate, Sodium phosphate dibasic, Repone K by formula ratio, said components is dissolved in the deionized water of formula ratio, filters and get final product.
Embodiment 2:
Improve a cultural method for duck flavivirus virus titre, it comprises the following steps:
The preparation of A, DMEM nutrient solution: 13.4g DMEM dry powder is dissolved in 1000ml deionized water, be stirred to after dissolving completely, adjust ph to 6.8, and carry out Sterile Filtration with 0.22um sterilised membrane filter, the refrigerator being placed in 4 DEG C is stand-by;
B, in above-mentioned DMEM solution, add the foetal calf serum accounting for DMEM nutrient solution volume 8% after, inoculate 293 cells and carry out monolayer culture;
C, after individual layer 293 cell cultures is good, discards the DMEM nutrient solution in culture vessel, and wash 3 times with sterile PBS buffer;
D, discard sterile PBS buffer, in culture vessel, then add the DMEM nutrient solution described in affinity agent and steps A, wherein the volumetric usage of affinity agent is 0.5% of DMEM nutrient solution volumetric usage;
E, culture vessel is placed in cell culture incubator quiescent culture 30min after, inoculation duck flavivirus, mix be placed in cell culture incubator cultivate 5 days, results virus liquid.
Particularly, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0.8%; Phosphatase 11 %;
Sorbic Acid 0.3%; Oxysuccinic acid 0.4%;
Inorganic salt 2.5%; Deionized water 95%.
Wherein, described inorganic salt are the mixing of sodium sulfate, Sodium phosphate dibasic, Repone K, sodium-chlor.
Wherein, described sodium sulfate accounts for 0.25% of affinity agent total amount; Described SODIUM PHOSPHATE, MONOBASIC accounts for 1% of affinity agent total amount; Described Repone K accounts for 0.5% of affinity agent total amount; Described sodium-chlor accounts for 0.75% of affinity agent total amount.
Particularly, described affinity agent is prepared from according to the following steps: take propanedioic acid, phosphoric acid, Sorbic Acid, oxysuccinic acid, sodium sulfate, Sodium phosphate dibasic, Repone K, sodium-chlor by formula ratio, said components is dissolved in the deionized water of formula ratio, filters and get final product.
Embodiment 3:
Improve a cultural method for duck flavivirus virus titre, it comprises the following steps:
The preparation of A, DMEM nutrient solution: 13.4g DMEM dry powder is dissolved in 1000ml deionized water, be stirred to after dissolving completely, adjust ph to 7.4, and carry out Sterile Filtration with 0.22um sterilised membrane filter, the refrigerator being placed in 4 DEG C is stand-by;
B, in above-mentioned DMEM solution, add the foetal calf serum accounting for DMEM nutrient solution volume 6% after, inoculation ST cell carries out monolayer culture;
C, after individual layer ST cell cultures is good, discard the DMEM nutrient solution in culture vessel, and wash 2 times with aseptic serum-free DMEM nutrient solution;
D, discard aseptic serum-free DMEM nutrient solution, in culture vessel, then add the DMEM nutrient solution described in affinity agent and steps A, wherein the volumetric usage of affinity agent is 0.8% of DMEM nutrient solution volumetric usage;
E, culture vessel is placed in cell culture incubator quiescent culture 30min after, inoculation duck flavivirus, mix be placed in cell culture incubator cultivate 4 days, results virus liquid.
Particularly, the affinity agent described in step D is prepared from by following each component by mass percentage:
Propanedioic acid 0.5%; Phosphoric acid 0.8%;
Sorbic Acid 0.2%; Oxysuccinic acid 0.5%;
Inorganic salt 2.7%; Deionized water 95.3%.
Wherein, described inorganic salt are the mixing of sodium sulfate, Sodium phosphate dibasic, sodium-chlor.
Wherein, described sodium sulfate accounts for 0.2% of affinity agent total amount; Described SODIUM PHOSPHATE, MONOBASIC accounts for 1.5% of affinity agent total amount; Described sodium-chlor accounts for 1% of affinity agent total amount.
Particularly, described affinity agent is prepared from according to the following steps: take propanedioic acid, phosphoric acid, Sorbic Acid, oxysuccinic acid, sodium sulfate, Sodium phosphate dibasic, sodium-chlor by formula ratio, said components is dissolved in the deionized water of formula ratio, filters and get final product.
Comparative example 1:
Technical solutions according to the invention are adopted to cultivate duck flavivirus, be divided into A group and B group, substratum and other culture condition identical, wherein A group does not add affinity agent, B group adds affinity agent, the virus of cultivation after 3 days is taken out then multigelation three times, and carry out chicken embryo medium lethal dose ELD
50measure.Specific as follows:
One, the acquisition of duck flavivirus liquid:
The preparation of A, DMEM nutrient solution: DMEM dry powder is dissolved in deionized water, be stirred to after dissolving completely, adjust ph to 7.4, carry out Sterile Filtration with 0.22um sterilised membrane filter and be divided into A group, B group, the refrigerator being placed in 4 DEG C is stand-by;
B, in the DMEM solution of A group, B group, add the foetal calf serum accounting for DMEM nutrient solution volume 6% respectively after, inoculate ST cell respectively and carry out monolayer culture;
C, after the individual layer ST cell cultures of A group, B group is good, discard the DMEM nutrient solution in culture vessel, wash 2 times with aseptic serum-free DMEM nutrient solution respectively;
D, discard aseptic serum-free DMEM nutrient solution, in A group culture vessel, then add the DMEM nutrient solution described in steps A; In B group culture vessel, add the DMEM nutrient solution described in affinity agent and steps A, wherein the volumetric usage of affinity agent is 0.8% of DMEM nutrient solution volumetric usage;
E, the culture vessel of A group, B group is placed in cell culture incubator quiescent culture 30min after, inoculation duck flavivirus, mixes to be placed in cell culture incubator and cultivates 4 days, the virus liquid of results A group, B group.
Wherein, the affinity agent added by B group is prepared from by following each component by mass percentage:
Propanedioic acid 0.5%; Phosphoric acid 0.8%;
Sorbic Acid 0.2%; Oxysuccinic acid 0.5%;
Inorganic salt 2.7%; Deionized water 95.3%.
Wherein, described inorganic salt are the mixing of sodium sulfate, Sodium phosphate dibasic, sodium-chlor.
Wherein, described sodium sulfate accounts for 0.2% of affinity agent total amount; Described SODIUM PHOSPHATE, MONOBASIC accounts for 1.5% of affinity agent total amount; Described sodium-chlor accounts for 1% of affinity agent total amount.
Wherein, described affinity agent is prepared from according to the following steps: take propanedioic acid, phosphoric acid, Sorbic Acid, oxysuccinic acid, sodium sulfate, Sodium phosphate dibasic, sodium-chlor by formula ratio, said components is dissolved in the deionized water of formula ratio, filters and get final product.
Two, duck virus ELD
50mensuration:
Step a, by centrifugal for the virus liquid of A group, B group, rotating speed is 8000r/min, and the time is 20min, gets supernatant, and uses 0.22um membrane filtration, obtains the viral supernatant liquid of A group, B group;
Step b, the viral supernatant liquid of above-mentioned A group, B group done in centrifuge tube the dilution of continuous 10 times, from 10
-1-10
-10;
Step c, the hatching SPF chicken embryo of 8 days is carried out disinfection stand-by;
Steps d, be inoculated in chick embryo allantoic cavity by the viral supernatant liquid of the A group of having diluted, B group, each dilution viral supernatant liquid is seeded in 6 pieces of chicken embryos respectively, every hole inoculation 100ul;
Step e, cultivation 5 days, and observe day by day and record result, result is as shown in table 1;
Step f, carry out calculating ELD by Karber method
50.
The dead chicken embryo ratio of table 1 A group, B group
| Virus liquid extent of dilution | There is the ratio of dead chicken embryo in A group | There is the ratio of dead chicken embryo in B group |
| 10 -1 | 5/6＝0.833 | 6/6＝1 |
| 10 -2 | 1/6＝0.166 | 6/6＝1 |
| 10 -3 | 0/6＝0 | 6/6＝1 |
| 10 -4 | 0/6＝0 | 5/6＝0.833 |
| 10 -5 | 0/6＝0 | 2/6＝0.333 |
| 10 -6 | 0/6＝0 | 2/6＝0.333 |
| 10 -7 | 0/6＝0 | 0/6＝0 |
| 10 -8 | 0/6＝0 | 0/6＝0 |
| 10 -9 | 0/6＝0 | 0/6＝0 |
1, method of calculation: lgELD
50=L-D (S-0.5)
Wherein, L is most high dilution logarithm; D is the difference between extent of dilution logarithm; S: positive boring ratio rate summation
2, calculation result:
According to table 1, the positive boring ratio rate summation of A group and B group is respectively: S
a=1, S
b=4.5
A group: lgELD
50=-1-(+1) * (1-0.5)=-1.5, then ELD
50=10
1.5/ 0.1ml
B group: lgELD
50=-1-(+1) * (4.5-0.5)=-5, then ELD
50=10
5/ 0.1ml
3, interpretation of result:
From table 1 and above-mentioned calculation result, with the addition of the ratio of the dead chicken embryo of B group appearance that affinity agent carries out cultivating much larger than the ratio not adding the dead chicken embryo of A group appearance that affinity agent carries out cultivating, similarly, the ELD of B group
50also much larger than the ELD of A group
50, illustrate that affinity agent of the present invention makes the virus titer of duck flavivirus be improved greatly; In addition, from the dead chicken embryo ratio result of B group, the duck flavivirus liquid of B group is diluted to 10
-6times time still there is certain virulence, and the duck flavivirus liquid of A group is only diluted to 10
-3doubly chicken embryo just can not be made lethal, can demonstrate,prove thus, cultural method of the present invention can improve the virus titer of duck flavivirus greatly.
Comparative example 2:
Hatching 36 pieces of SPF chicken embryos of 8 days are carried out disinfection and is equally divided into three groups of positive controls and three groups of negative control group are stand-by, every piece of chicken embryo of three groups of negative control group all inoculates the ST cell culture fluid of 100ul, three groups of positive controls inoculate the 100ul duck flavivirus stoste of embodiment 1 ~ 3 gained respectively, and result is as shown in table 2;
The dead chicken embryo ratio of table 2 negative control group, positive controls
| Embodiment | There is the ratio of dead chicken embryo in negative control group | There is the ratio of dead chicken embryo in positive controls |
| 1 | 0/6＝0 | 6/6＝1 |
| 2 | 0/6＝0 | 6/6＝1 |
| 3 | 0/6＝0 | 6/6＝1 |
Interpretation of result:
The duck flavivirus adopted in comparative example 2 is inoculated in ST cell culture fluid can make the chicken embryo of all this virus of inoculation in experimental group occur death, and multiplicity is high, illustrates that affinity agent of the present invention makes the virus titer of duck flavivirus be improved greatly.Adopt the cultural method used in the present invention can improve duck flavivirus titre 10-100 doubly.
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (9)
1. improve a cultural method for duck flavivirus virus titre, it is characterized in that: it comprises the following steps:
The preparation of A, DMEM nutrient solution: be dissolved in deionized water by DMEM dry powder, is stirred to after dissolving completely, adjust ph to 6.8 ~ 7.8, and carry out Sterile Filtration with the sterilised membrane filter of≤0.22 μm, be placed in≤refrigerator of 4 DEG C is stand-by;
B, in above-mentioned DMEM solution, add the foetal calf serum accounting for DMEM nutrient solution volume 1% ~ 8% after, inoculating cell carries out monolayer culture;
C, after monolayer cell culture is good, discards the DMEM nutrient solution in culture vessel, and wash by aseptic washing lotion;
D, discard aseptic washing lotion, in culture vessel, then add the DMEM nutrient solution described in affinity agent and steps A, wherein the volumetric usage of affinity agent is 0.01% ~ 1% of DMEM nutrient solution volumetric usage;
E, culture vessel is placed in cell culture incubator quiescent culture 1min ~ 30min after, inoculation duck flavivirus, mix be placed in cell culture incubator cultivate 2 ~ 5 days, results virus liquid.
2. cultural method according to claim 1, is characterized in that: the cell described in step B is the one in Vero cell, 293 cells, ST cell, bhk cell.
3. cultural method according to claim 1, is characterized in that: the aseptic washing lotion described in step C is the one in deionized water, PBS damping fluid, serum-free DMEM nutrient solution.
4. cultural method according to claim 1, is characterized in that: the affinity agent described in step D is prepared from by following each component by mass percentage:
5. cultural method according to claim 4, is characterized in that: the affinity agent described in step D is prepared from by following each component by mass percentage:
6. cultural method according to claim 4, is characterized in that: the affinity agent described in step D is prepared from by following each component by mass percentage:
7. cultural method according to claim 4, is characterized in that: described inorganic salt are one or more in sodium sulfate, Sodium phosphate dibasic, Repone K, sodium-chlor.
8. cultural method according to claim 7, is characterized in that: described sodium sulfate accounts for 0.15 ~ 0.3% of affinity agent total amount; Described SODIUM PHOSPHATE, MONOBASIC accounts for the 0.1-1.6% of affinity agent total amount; Described sodium-chlor accounts for the 0.09-0.9% of affinity agent total amount; Described Repone K accounts for the 0.01-1% of affinity agent total amount.
9. the cultural method according to any one of claim 4 ~ 6, it is characterized in that: described affinity agent is prepared from according to the following steps: take propanedioic acid, phosphoric acid, Sorbic Acid, oxysuccinic acid, inorganic salt by formula ratio, said components is dissolved in the deionized water of formula ratio, filters and get final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510050831.4A CN104560892B (en) | 2015-01-31 | 2015-01-31 | Culture method for increasing virus titer of duck flavivirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510050831.4A CN104560892B (en) | 2015-01-31 | 2015-01-31 | Culture method for increasing virus titer of duck flavivirus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104560892A true CN104560892A (en) | 2015-04-29 |
| CN104560892B CN104560892B (en) | 2017-01-18 |
Family
ID=53077972
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510050831.4A Active CN104560892B (en) | 2015-01-31 | 2015-01-31 | Culture method for increasing virus titer of duck flavivirus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104560892B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624123A (en) * | 2016-03-29 | 2016-06-01 | 四川海林格生物制药有限公司 | Cell affinity agent for improving porcine pseudorabies virus in-vitro infection efficiency and use method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220290A (en) * | 2011-05-25 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Cell culture method for duck flavivirus |
| CN103143009A (en) * | 2013-03-07 | 2013-06-12 | 齐鲁动物保健品有限公司 | Method for producing duck tembusu virus inactivated vaccines in large scale |
-
2015
- 2015-01-31 CN CN201510050831.4A patent/CN104560892B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220290A (en) * | 2011-05-25 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Cell culture method for duck flavivirus |
| CN103143009A (en) * | 2013-03-07 | 2013-06-12 | 齐鲁动物保健品有限公司 | Method for producing duck tembusu virus inactivated vaccines in large scale |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624123A (en) * | 2016-03-29 | 2016-06-01 | 四川海林格生物制药有限公司 | Cell affinity agent for improving porcine pseudorabies virus in-vitro infection efficiency and use method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104560892B (en) | 2017-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103074246B (en) | A kind of low serum high-efficient culture chicken virus mycoplasma low virulent strain substratum and preparation method thereof | |
| CN102816732B (en) | MDCK cell line suitable for full suspended serum-free culture and its application in culturing of influenza virus and production of influenza virus vaccines | |
| CN104001167A (en) | Process for preparing avian influenza inactivated vaccine by full suspended culture cells and product | |
| CN103060220A (en) | Low-serum culture medium for efficiently culturing mycoplasma hyopneumoniae and preparation method thereof | |
| CN102690791A (en) | Method for generating porcine pseudorabies virus by culturing ST cell in microcarrier of bioreactor | |
| CN102127524B (en) | Method for proliferating avian influenza viruses in bioreactor with cell carrier | |
| CN103394081B (en) | WAVE wave bioreactor is utilized to produce the method for avian influenza vaccine | |
| CN102861329A (en) | Production method of canine parvovirus inactivated vaccine by utilizing bioreactor | |
| CN104338127A (en) | Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine | |
| CN103468649B (en) | Rapid adaptation method of rabies vaccine virus strains for human body on diploid cells of human body | |
| CN105198635A (en) | Macro-element nutrient solution for large-scale culture of Chlorella salina | |
| CN104611297B (en) | A kind of cultural method of Porcine epidemic diarrhea virus | |
| CN105838683A (en) | Method for proliferation of mink canine distemper virus by applying novel cell microcarrier | |
| CN101914485A (en) | Method for preparing serum-free soybean protein peptide animal cell medium | |
| CN104560892A (en) | Culture method for increasing virus titer of duck flavivirus | |
| CN104611298A (en) | Cell affinity agent capable of improving duck flavivirus in-vitro infection efficiency and preparation method of cell affinity agent | |
| CN102886043B (en) | Binary inactivated vaccine against Japanese encephalitis virus and porcine parvovirus and preparation method thereof | |
| CN105838641A (en) | Mycoplasma synoviae culture method | |
| CN105288610A (en) | Production technology and product of inactivated vaccine for avian influenza | |
| CN104630159A (en) | Method for producing hog cholera C-strain virus by culturing ST Cells in low serum | |
| CN104278010A (en) | Serum-free chemical culture medium for MDCK cell | |
| CN103865885A (en) | Culture method of porcine parvovirus | |
| CN109679900B (en) | Preparation method of avian influenza vaccine and product thereof | |
| CN103536913A (en) | Method for producing avian influenza virus bivalent inactivated vaccine by use of MDCK cell line | |
| CN104046588A (en) | Micro-carrier culture method of bioreactor of MDCK (Madin Darby Canine Kidney) cells and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20161205 Address after: 511490 Guangdong province Panyu District Shatou street Guangzhou City Fu Ping Lu eight Street 5 (only for office use) Applicant after: Guangdong Haid Animal Husbandry Veterinary Research Institute Co., Ltd. Applicant after: SICHUAN HAILINGE BIOLOGICAL PHARMACEUTICAL CO., LTD. Address before: 511490 Guangdong province Panyu District Shatou street Guangzhou City Fu Ping Lu eight Street 5 (only for office use) Applicant before: Guangdong Haid Animal Husbandry Veterinary Research Institute Co., Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |