CN104450630A - Method for culturing goose parvovirus by using goose embryo fibroblast line - Google Patents
Method for culturing goose parvovirus by using goose embryo fibroblast line Download PDFInfo
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- CN104450630A CN104450630A CN201410710792.1A CN201410710792A CN104450630A CN 104450630 A CN104450630 A CN 104450630A CN 201410710792 A CN201410710792 A CN 201410710792A CN 104450630 A CN104450630 A CN 104450630A
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Abstract
The invention provides a method for culturing goose parvovirus by using a goose embryo fibroblast line, belongs to the field of veterinary biological products, and solves the problem of low production efficiency of an existing goose parvovirus culturing method. The method comprises the following steps: culturing primary cells, namely cutting a goose embryo which well develops into small pieces, washing, digesting, blowing and dispersing digestive cells to obtain a cell suspension, culturing until the cells form complete monolayers so as to obtain the primary cells; establishing a goose embryo fibroblast line, namely removing a nutrient solution from the primary cells of the goose embryo fibroblast, washing, digesting and culturing until complete monolayer cells are formed to obtain F1-generation cells which can be passed by three generations; reproducing and harvesting, namely inoculating F1-F4-generation cells which form 70 percent of monolayer, and harvesting reproduced viruses when over 75 percent of cells is diseased. According to the method, the amount of harvested virus liquid is 255 times of the dose of primary cell inoculation culturing, and the virus titer of the virus liquid is 10-100 times higher than that of a virus liquid which is inoculated, cultured and harvested when 100 percent of monolayer is formed.
Description
Technical field
The invention belongs to veterinary biologics field, be specifically related to a kind of cultural method of Goose Parvovirus.
Background technology
Goose industry development in recent years is supported rapidly by China, and the number of animals raised increases year by year, in increasing peasant income and industry restructuring, serve active promoting function.Goose product be it is believed that it is pure green food, and its byproduct economic worth is also very high, and market has openings is large, and economic benefit is obvious, and these conditions further facilitate the fast development of foster goose industry.But ensure that foster goose industry is stable to develop in a healthy way, a set of science, effective control and prevention of disease measure must be set up.
Gosling plague (GP) is that the Important Infectious Diseases of goose industry is supported in harm at present, and this sick provisions goose field (family) causes serious financial loss, is also that one of principal element of goose industry fast development is supported in restriction simultaneously.Gosling plague is the acute or subacute septic transmissible disease of young goose caused by goose parvovirus (GPV) (being Goose Parvovirus again), this disease mainly encroaches on out the young goose of 4 ~ 20 ages in days after shell, have and propagate fast, sickness rate and the high feature of lethality rate, mortality ratio can reach 90% ~ 100%, along with young goose age in days increases sickness rate and lethality rate decline.Fang Dingyi (1956) first finds this disease in China, after nineteen sixty-five, the country such as Hungary, Germany, Holland, USSR (Union of Soviet Socialist Republics), Italy, Britain, France, Yugoslavia, Vietnam, Israel successively reports the existence of this disease, and there is breaking out with popular of this disease in the area of the current world many raisings goose and kind duck.
At present, the effective measure of prevention gosling plague are the immunizations of gosling plague bacterin.China successfully develops kind of goose and young goose duck embryo or goose embryo attenuated vaccine in succession, and effectively controls the breaking out with popular of this disease.But we find in production practice in recent years; existing gosling plague bacterin production method efficiency is lower; be unfavorable for large-scale production; its major cause is that existing method is with duck embryo, goose embryo or primary fibroblast virus of proliferation; due to duck embryo, goose embryo or primary cell large usage quantity, the cycle of hatching is longer, and production technique is relatively loaded down with trivial details; thus cause the viral proliferation amount of unit goose embryo limited, the culture efficiency of virus is low.
Summary of the invention
The object of the invention is to solve the lower problem of existing Goose Parvovirus method of cultivation production efficiency, and a kind of method of cultivating Goose Parvovirus with goose embryo fibroblast is provided.
These concrete steps of cultivating the method for Goose Parvovirus with goose embryo fibroblast are as follows:
(1) cultivation of goose embryo fibroblast primary cell: get the well-developed goose embryo of 12 age in days, with iodine tincture cotton and alcohol swab, sterilized in eggshell air chamber position, aseptic taking-up goose embryo, to decaptitate four limbs and internal organ, put into the glass dish of sterilizing, with DMEM solution washing idiosome three times, the large small tissue blocks of the grain of rice is cut into the scissors of sterilizing, 2 times are washed again with DMEM solution, then 0.25% trypsin solution (each goose embryo adds 3 ~ 5ml) is added, 5 ~ 10min is digested in 37.5 DEG C of water-baths, sucking-off trypsin solution, with DMEM solution washing 2 times, then add containing 8% calf serum, the DMEM solution piping and druming dispersion peptic cell of pH value 7.0 ~ 7.5, every milliliter of cell suspension containing viable count 100 ~ 1,500,000 is obtained after filtration, be sub-packed in culturing bottle, 37 DEG C of constant temperature static gas wave refrigerator, after cell forms intact monolayer, obtain goose embryo fibroblast primary cell,
(2) foundation of goose embryo fibroblast: the goose embryo fibroblast primary cell obtained in step () is discarded nutrient solution, add PBS buffer solution 2 times, sucking-off washing lotion, then 0.25% trypsin solution is added, cultivate digestion 3 ~ 5min for 37.5 DEG C, when monolayer cell draws sky, edge to depart from bottle wall, sucking-off Digestive system, adds nutritive medium and piping and druming dispersion peptic cell; Add nutritive medium by the cell concn of 80 ~ 1,000,000/ml, be sub-packed in culturing bottle and cultivate, 37 DEG C of constant temperature static gas wave refrigerator, to forming intact monolayer cell, namely obtain F1 generation cell; Then F1 generation cell is continued Secondary Culture, can pass for 3 generations, obtain F1 ~ F4 for cell; Described nutritive medium is the DMEM solution containing 8% calf serum, pH value 7.0 ~ 7.5;
(3) breeding of Goose Parvovirus and results: the goose embryo fibroblast F4 substitute that step (two) is cultivated is in viral proliferation, the F4 of formation 70% individual layer is discarded nutrient solution for cell, by the inoculum size access Goose Parvovirus seed culture of viruses of square vase in 1ml/, 1h is cultivated in 37 DEG C of absorption, then viruses adsorption liquid is discarded, in 10ml/, the amount of square vase adds nutritive medium, 37 DEG C of constant temperature static gas wave refrigerator 72 ~ 96 hours, when there is pathology in more than 75% cell, stop cultivating, after virus-culturing fluid multigelation three times, sterile collection cell culture fluid; Described nutritive medium is the DMEM solution containing 8% calf serum, pH value 7.0 ~ 7.5.
Goose embryo fibroblast primary cell or F1 ~ F4 when being cultured to cell and forming 70% individual layer, all can be used for the breeding of Goose Parvovirus for the cell of any generation in cell.
The present invention has following beneficial effect:
The method of cultivation Goose Parvovirus of the present invention is to set up based on goose embryo fibroblast, can by goose embryo fibroblast primary cell culture to F4 generation by the foundation of goose embryo fibroblast, with goose embryo fibroblast F4 for cell inoculation culture virus, the viral liquid measure of its results is 255 times that primary cell connects malicious cultivation amount, thus substantially increase virus culture efficiency, can ensure that virus has stable immunogenicity simultaneously.The method of cultivation Goose Parvovirus of the present invention adopts to the step of cell access seed culture of viruses carries out Goose Parvovirus and connects poison cultivation when cell forms 70% individual layer, after measured, the virus liquid poison valency connecing poison cultivation results when adopting the viral suspension poison valency of aforesaid method results to form 100% individual layer compared with cell is high 10 ~ 100 times.The method of cultivation Goose Parvovirus of the present invention is except available F4 is for except passage cell cultivation virus, in goose embryo fibroblast succeeding generations, primary cell and F1 ~ F3 all can be used for cultivating virus for any generation cell in cell, experimenter can experimental progress of work arrangement, the cell taking out formation 70% individual layer from above-mentioned any generation cell carries out connecing poison, thus can experiment arrangement process flexibly.
Embodiment
These concrete steps of cultivating the method for Goose Parvovirus with goose embryo fibroblast are as follows:
(1) cultivation of goose embryo fibroblast primary cell: get the well-developed goose embryo of 12 age in days, with iodine tincture cotton and alcohol swab, sterilized in eggshell air chamber position, aseptic taking-up goose embryo, to decaptitate four limbs and internal organ, put into the glass dish of sterilizing, with DMEM solution washing idiosome three times, the large small tissue blocks of the grain of rice is cut into the scissors of sterilizing, 2 times are washed again with DMEM solution, then 0.25% trypsin solution (each goose embryo adds 3 ~ 5ml) is added, 5 ~ 10min is digested in 37.5 DEG C of water-baths, sucking-off trypsin solution, with DMEM solution washing 2 times, then add containing 8% calf serum, the DMEM solution piping and druming dispersion peptic cell of pH value 7.0 ~ 7.5, every milliliter of cell suspension containing viable count 100 ~ 1,500,000 is obtained after filtration, be sub-packed in culturing bottle, 37 DEG C of constant temperature static gas wave refrigerator, after cell forms intact monolayer, obtain goose embryo fibroblast primary cell,
(2) foundation of goose embryo fibroblast: the goose embryo fibroblast primary cell obtained in step () is discarded nutrient solution, add PBS buffer solution 2 times, sucking-off washing lotion, then 0.25% trypsin solution is added, cultivate digestion 3 ~ 5min for 37.5 DEG C, when monolayer cell draws sky, edge to depart from bottle wall, sucking-off Digestive system, adds nutritive medium and piping and druming dispersion peptic cell; Add nutritive medium by the cell concn of 80 ~ 1,000,000/ml, be sub-packed in culturing bottle and cultivate, 37 DEG C of constant temperature static gas wave refrigerator, to forming intact monolayer cell, namely obtain F1 generation cell; Then F1 generation cell is continued Secondary Culture, can pass for 3 generations, obtain F1 ~ F4 for cell; Described nutritive medium is the DMEM solution containing 8% calf serum, pH value 7.0 ~ 7.5;
(3) breeding of Goose Parvovirus and results: goose embryo fibroblast F1 ~ F4 substitute that step (two) is cultivated is in viral proliferation, F1 ~ the F4 of formation 70% individual layer is discarded nutrient solution for cell, by the inoculum size access Goose Parvovirus seed culture of viruses of square vase in 1ml/, 1h is cultivated in 37 DEG C of absorption, then viruses adsorption liquid is discarded, in 10ml/, the amount of square vase adds nutritive medium, 37 DEG C of constant temperature static gas wave refrigerator 72 ~ 96 hours, when there is pathology in more than 75% cell, results propagative viruses, by virus culture bottle multigelation three times, sterile collection cell culture fluid; Described nutritive medium is the DMEM solution containing 8% calf serum, pH value 7.0 ~ 7.5.This step adopt Goose Parvovirus seed culture of viruses be Goose Parvovirus (
goose parvovirus) ATCC SHM 319, this seed culture of viruses is preserved in American Type Culture Collecti (ATCC), and preserving number ATCC VR-696, can buy in American Type Culture Collecti.
This step receives the cell culture fluid poison valency (TCID of poison time to difference
50) measure, particularly: use TCID
50measuring method measures the malicious valency (TCID of 48h ~ 72h, 72h ~ 96h, 96h ~ 120h harvested cell nutrient solution respectively
50).TCID
50measuring method: 24 orifice plate goose embryos become clone primary cell for cell cultures, are used for connecing poison when 20h forms 70% individual layer.Harvested cell nutrient solution is done 10 times of doubling dilutions, get 10
-6, 10
-7, 10
-8, 10
-9four extent of dilution inoculation culture, 0.1ml/ hole, often dilution connects four holes.1h are cultivated in 37 DEG C of absorption, after abandon adsorption liquid, add containing 3% calf serum DMEM nutritive medium, 37 DEG C of static gas wave refrigerator, 120h sentences eventually.Malicious valency is calculated by Reed and Muench Liang Shi method.Result shows, 48h ~ 72h harvested cell nutrient solution virus titer is minimum, and when incubation time is 72h ~ 96h, malicious valency is the highest, with 96h ~ 120h harvested cell nutrient solution poison valency no significant difference, finally determines that the best virus harvest time should at 96h.Poison valency measurement result is in table 1.
Table 1
| Virus liquid classification | 48h~72h | 72h~96h | 96h~120h |
| Poison valency TCID 50/ml | 10 -7.5 | 10 -8.3 | 10 -8 |
The present invention, except measuring the malicious valency of virus-culturing fluid, has also carried out following mensuration to the virus-culturing fluid of results:
(1) specific detection: virus-culturing fluid (200TCID will be gathered in the crops
50/ ml) mix with equivalent anti gosling plague specific serum, put in 37 DEG C of incubators and make 1h with sense, inoculation goose embryo fibroblast individual layer, cultivate and observe 96h, monolayer cell does not produce cytopathy (CPE).
(2) pure property detects: test by " People's Republic of China's veterinary drug allusion quotation " annex, detected result: pollute without bacterium, mould, mycoplasma, exogenous virus.
The present invention also to Goose Parvovirus (
parvovirus Goose parvovirus) CVCC AV240, Goose Parvovirus (
parvovirus Goose parvovirus) C CVCC AV239, Goose Parvovirus (
parvovirus Goose parvovirus) C NEAU0671 tri-Goose Parvovirus seeds culture of viruses adopt aforesaid method of the present invention to cultivate, experimental result shows, 70% of above-mentioned three seeds culture of viruses connect the final nutrient solution viral level of poison and are respectively 10
8.2tCID
50/ ml, 10
8.4tCID
50/ ml, 10
8.5tCID
50/ ml, 100% connects the final nutrient solution viral level of poison is respectively 10
6.1tCID
50/ ml, 10
6tCID
50/ ml, 10
6.2tCID
50/ ml, thus adopts method of the present invention to cultivate above-mentioned three seeds culture of viruses and all can reach the culture effect identical with Goose Parvovirus ATCC SHM 319.Above-mentioned three seeds culture of viruses are all deposited in National Veterinary Microbiological Culture Collection administrative center (CVCC), all buy by this preservation mechanism.
The present invention compares test to the goose embryo fibroblast of formation 70% individual layer and the goose embryo fibroblast forming 100% intact monolayer for cultivating Goose Parvovirus, and concrete steps are as follows:
(1) goose embryo fibroblast is cultured to formation 70% individual layer, discard nutrient solution, by the inoculum size access Goose Parvovirus seed culture of viruses of square vase in 1ml/, 1h is cultivated in 37 DEG C of absorption, then discard viruses adsorption liquid, add nutritive medium by the amount of square vase in 10ml/, 37 DEG C of constant temperature static gas wave refrigerator 72 ~ 96 hours, when there is pathology in more than 75% cell, results propagative viruses.By virus culture bottle multigelation three times, sterile collection cell culture fluid ,-20 DEG C of preservations.Leave and take sample simultaneously, detect for viral level.
(1) goose embryo fibroblast is cultured to formation 100% intact monolayer, discard nutrient solution, by the inoculum size access Goose Parvovirus seed culture of viruses of square vase in 1ml/, 1h is cultivated in 37 DEG C of absorption, then discard viruses adsorption liquid, add nutritive medium by the amount of square vase in 10ml/, 37 DEG C of constant temperature static gas wave refrigerator 72 ~ 96 hours, when there is pathology in more than 75% cell, results propagative viruses.By virus culture bottle multigelation three times, sterile collection cell culture fluid ,-20 DEG C of preservations.Leave and take sample simultaneously, detect for viral level.
(3) viral level detects: adopt TCID
50measuring method detects: above two samples are done 10 times of doubling dilutions respectively, gets 10
-6, 10
-7, 10
-8, 10
-9four extent of dilution inoculation culture, 0.1ml/ hole, often dilution connects four holes, and 1h are cultivated in 37 DEG C of absorption, after abandon adsorption liquid, add containing 3% calf serum DMEM nutritive medium, 37 DEG C of static gas wave refrigerator, 120h sentences eventually.Malicious valency is calculated by Reed and Muench Liang Shi method.
Result shows, and adopt the goose embryo fibroblast of 70% individual layer to cultivate the gosling blast venom of breeding, viral level is TCID
50﹦ 10
-8.3/ ml, adopt the goose embryo fibroblast of 100% intact monolayer to cultivate the gosling blast venom of breeding, viral level is TCID
50﹦ 10
-6.1/ ml.Test-results shows: connect when adopting goose embryo fibroblast to form 70% individual layer and to connect poison when virus liquid viral level that poison cultivates results forms 100% individual layer compared with goose embryo fibroblast to cultivate the virus liquid viral level of results high 100 times.
In method of the present invention, goose embryo fibroblast primary cell or F1 ~ F3 when being cultured to cell and forming 70% individual layer, all can be used for the breeding of Goose Parvovirus for the cell of any generation in cell.Except available F4 cultivates except virus for passage cell, in goose embryo fibroblast succeeding generations, primary cell and F1 ~ F3 all can be used for cultivating virus for any generation cell in cell, experimenter can experimental progress of work arrangement, the cell taking out formation 70% individual layer from above-mentioned any generation cell carries out connecing poison, thus can experiment arrangement process flexibly.
Claims (2)
1. cultivate the method for Goose Parvovirus with goose embryo fibroblast, it is characterized in that: the concrete steps of the method are as follows:
(1) cultivation of goose embryo fibroblast primary cell: get the well-developed goose embryo of 12 age in days, with iodine tincture cotton and alcohol swab, sterilized in eggshell air chamber position, aseptic taking-up goose embryo, to decaptitate four limbs and internal organ, put into the glass dish of sterilizing, with DMEM solution washing idiosome three times, the large small tissue blocks of the grain of rice is cut into the scissors of sterilizing, 2 times are washed again with DMEM solution, then 0.25% trypsin solution (each goose embryo adds 3 ~ 5ml) is added, 5 ~ 10min is digested in 37.5 DEG C of water-baths, sucking-off trypsin solution, with DMEM solution washing 2 times, then add containing 8% calf serum, the DMEM solution piping and druming dispersion peptic cell of pH value 7.0 ~ 7.5, every milliliter of cell suspension containing viable count 100 ~ 1,500,000 is obtained after filtration, be sub-packed in culturing bottle, 37 DEG C of constant temperature static gas wave refrigerator, after cell forms intact monolayer, obtain goose embryo fibroblast primary cell,
(2) foundation of goose embryo fibroblast: the goose embryo fibroblast primary cell obtained in step () is discarded nutrient solution, add PBS buffer solution 2 times, sucking-off washing lotion, then 0.25% trypsin solution is added, cultivate digestion 3 ~ 5min for 37.5 DEG C, when monolayer cell draws sky, edge to depart from bottle wall, sucking-off Digestive system, adds nutritive medium and piping and druming dispersion peptic cell; Add nutritive medium by the cell concn of 80 ~ 1,000,000/ml, be sub-packed in culturing bottle and cultivate, 37 DEG C of constant temperature static gas wave refrigerator, to forming intact monolayer cell, namely obtain F1 generation cell; Then F1 generation cell is continued Secondary Culture, can pass for 3 generations, obtain F1 ~ F4 for cell; Described nutritive medium is the DMEM solution containing 8% calf serum, pH value 7.0 ~ 7.5;
(3) breeding of Goose Parvovirus and results: goose embryo fibroblast F1 ~ F4 substitute that step (two) is cultivated is in viral proliferation, F1 ~ the F4 of formation 70% individual layer is discarded nutrient solution for cell, by the inoculum size access Goose Parvovirus seed culture of viruses of square vase in 1ml/, 1h is cultivated in 37 DEG C of absorption, then viruses adsorption liquid is discarded, in 10ml/, the amount of square vase adds nutritive medium, 37 DEG C of constant temperature static gas wave refrigerator 72 ~ 96 hours, when there is pathology in more than 75% cell, results propagative viruses, by virus culture bottle multigelation three times, sterile collection cell culture fluid; Described nutritive medium is the DMEM solution containing 8% calf serum, pH value 7.0 ~ 7.5.
2. method of cultivating Goose Parvovirus with goose embryo fibroblast according to claim 1, it is characterized in that: goose embryo fibroblast primary cell or F1 ~ F4 when being cultured to cell and forming 70% individual layer, all can be used for the breeding of Goose Parvovirus for the cell of any generation in cell.
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Cited By (3)
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| CN108949672A (en) * | 2018-08-02 | 2018-12-07 | 重庆市畜牧科学院 | Goose fibroblast culture medium and preparation method thereof |
| CN109125354A (en) * | 2018-10-31 | 2019-01-04 | 郭建德 | It is a kind of for eliminating or inhibiting the composition of carcinoma and soft tissue hyperplasia body |
| CN114381435A (en) * | 2022-01-19 | 2022-04-22 | 杨凌绿方生物工程有限公司 | Method for culturing H9N2 subtype avian influenza virus by using CEF spinner bottle |
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2014
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| CN104152403A (en) * | 2014-08-19 | 2014-11-19 | 山东省滨州畜牧兽医研究院 | Method for establishing goose embryo epithelial cell line and established goose embryo epithelial cell line |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949672A (en) * | 2018-08-02 | 2018-12-07 | 重庆市畜牧科学院 | Goose fibroblast culture medium and preparation method thereof |
| CN109125354A (en) * | 2018-10-31 | 2019-01-04 | 郭建德 | It is a kind of for eliminating or inhibiting the composition of carcinoma and soft tissue hyperplasia body |
| CN114381435A (en) * | 2022-01-19 | 2022-04-22 | 杨凌绿方生物工程有限公司 | Method for culturing H9N2 subtype avian influenza virus by using CEF spinner bottle |
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Application publication date: 20150325 |