CN102628030B - Chicken embryo culture method of Portunus tritubereulatus reovirus - Google Patents
Chicken embryo culture method of Portunus tritubereulatus reovirus Download PDFInfo
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Abstract
The invention discloses a chicken embryo culture method of Portunus tritubereulatus reovirus and is characterized by comprising the following steps of: using SPF chicken embryo as an in vitro culture system of Portunus tritubereulatus reovirus, selecting healthy chicken embryo of 3 and 4 days-old, inoculating a prepared Portunus tritubereulatus reovirus inoculated solution onto a chorioallantoic membrane of the chicken embryo, continuously culturing until the chicken embryo is dead, collecting dead chicken embryo cultured for 3-10 days, separating the chorioallantoic membrane and amniotic fluid, mixing the chorioallantoic membrane and the amniotic fluid, homogenizing the mixture, centrifuging and filtering to obtain a supernatant of Portunus tritubereulatus reovirus. The invention has the following advantages: a more efficient and adaptive Portunus tritubereulatus reovirus in vitro culture method is provided for rapid and effective in vitro subcultring of the virus; and the method overcomes the defect that there is no adaptive culture system during the culture process of Portunus tritubereulatus reovirus, fills the blank of chicken embryo culture system of crab virus and has laid a foundation for further research and application.
Description
Technical field
The present invention relates to the extracorporeal culturing method of Portunus trituberculatus Miers reovirus, especially relate to the chicken embryo culture method of Portunus trituberculatus Miers reovirus.
Background technology
Reovirus is the very wide virus of a class host range, and its host can be contained Mammals, bird, insect, hydrocoles etc.Infect a hydrobiological class reovirus and be classified as Aquareovirus.Meyers equals to be separated to Aquareovirus first from U.S. oyster in 1979.The Aquareovirus of isolation identification has more than 40 strains at present.These viruses mainly infect the host by breathing and enteron aisle, cause the disease of host's hemorrhagic disease and liver and pancreas.Caused huge loss in culture fishery in the world, serious threat the development of culture fishery.Aquareovirus is ubiquitous virus in hydrocoles, and report is all arranged in fish, shrimp, crab.
Portunus trituberculatus Miers is the important aquaculture kind of China, and in the Jiangsu and Zhejiang Provinces of China, the ground such as Guangdong and Guangxi Provinces, Fujian have started successful large-scale cultivation, in recent years along with the expansion of cultivation scale, various diseases frequently break out.Seminar is in the research process to the Portunus trituberculatus Miers disease, first from the existence of reovirus having been detected in ill Portunus trituberculatus Miers body, test etc. and to determine it for the main pathogen of swimming crab mortality autumn and winter by the artificial challenge, this virus has been brought huge loss to the cultivation of Portunus trituberculatus Miers.In order to find the effective ways of prevention and control Portunus trituberculatus Miers reovirus infection, this virus is carried out to the aspect researchs such as Pathogen Biology, diagnostics, infection mechanism and seem particularly important.This just must have stable viral source, and the best way carries out vitro culture to it exactly.Up to now, both at home and abroad viral vitro culture is focused mostly on technical in cell cultures etc., although reovirus has wider host range, but difference between species is larger, therefore, utilizing cell cultures to carry out vitro culture to the Portunus trituberculatus Miers reovirus needs corresponding cell strain or clone, also there is no ripe crab cell culture technology at present.In addition, cell culture method exists cultivates the shortcomings such as cost is high, the culture condition requirement is high, also exists the problem that can virus go down to posterity simultaneously.
The chicken embryo culture is a kind of cultural method that is widely used in mammalian virus, bird virus etc.; as Granted publication number is CN101633911A; the day for announcing is on January 27th, 2010; denomination of invention is " cultural method that is suitable for virus in the purification technique of mass production of human-used avian influenza vaccine "; just adopted the 9-11 age in days to carry out virus culture without deformity, clear, the movable healthy chicken embryo of blood vessel, this cultural method can be large-scale production human-used avian influenza virolysis vaccine enough, qualified avian influenza virus is provided.But the cultivation of some higher animal viruses is more common in viral chicken embryo culture, extremely rare in the research application in Aquatic animals virus field.At present, both at home and abroad also not about utilizing chicken embryo culture method to cultivate the correlative study report that the crab viroid comprises the method for Portunus trituberculatus Miers reovirus.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of chicken embryo culture method of Portunus trituberculatus Miers reovirus, the method is applied to chick embryo technique the cultivation of Portunus trituberculatus Miers reovirus first, can realize that the Portunus trituberculatus Miers reovirus is quick, efficient, stable, subculture in vitro separately is cultivated easily.
The present invention solves the problems of the technologies described above adopted technical scheme:
A kind of reovirus, described virus is Portunus trituberculatus Miers reovirus (Portunnus trituberculatusreovirus), this virus is spherical symmetrical structure, without cyst membrane, diameter 30 ± 10nm.Strain was the R1015 strain, and preserving number is CGMCC No.5379, was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 21st, 2011.
A kind of chicken embryo culture method of Portunus trituberculatus Miers reovirus, adopt the culture system in vitro of SPF chicken embryo as the Portunus trituberculatus Miers reovirus, select 3-4 age in days healthy chicken embryo, previously prepared good Portunus trituberculatus Miers reovirus inoculation liquid is seeded on chick chorioallantoic membrane, continue to cultivate until the chicken embryo occurs dead, the dead chicken embryo that the collection incubation time is 3-10 days carries out homogenate, centrifugation, obtains the supernatant liquor containing the Portunus trituberculatus Miers reovirus.
Concrete operation steps is as follows:
(1) choose the fresh kind of egg that meets the SPF specification, upwards 38 ℃ of hatchings of air chamber, relative humidity 50%, every 4 hours egg-turnings once, cultivate after 3-4 days, choose blood vessel thick and be the good chicken embryo of cherry healthy development at inside-paint discharge chamber edge, darkroom and indicate the great vessels present position, use in order to virus inoculation;
(2) choose some of Portunus trituberculatus Miers that infect reovirus, get the gill, muscle and internal organ, in mass volume ratio 1g: the 10ml ratio joins 12 ‰ the TNE damping fluid that PH is 7.4, homogenate in homogenizer by the mixture of the gill, muscle and internal organ, by homogenate in 4 ℃, 6000g, after centrifugal 20min, get containing viral supernatant liquor, the precipitation of centrifugal gained is resuspended with 12 ‰ TNE of 1～2ml, homogenate again, by homogenate obtains again homogenate in 4 ℃, 6000g, centrifugal 20min, the supernatant liquor that merges the two times centrifugal gained, first with the filter membrane suction filtration of 0.45 μ m, remove fragment of tissue by merging the gained supernatant liquor, use again the filter membrane suction filtration degerming of 0.22 μ m, after every milliliter of supernatant liquor after degerming adds 1000 unit penicillin and 1000ug Streptomycin sulphate, in the processing of spending the night of 4 ℃ of ice baths, obtain Portunus trituberculatus Miers reovirus inoculation liquid,
(3) choose health, the chicken embryo of well-developed 3-4 age in days, inoculum size by Portunus trituberculatus Miers reovirus inoculation liquid by 0.15-0.2ml is inoculated on the allantois of chicken embryo, cultivate 13-14 days by having inoculated viral chicken embryo under the constant temperature of 32.5 ℃, the dead chicken embryo that the collection incubation time is 3-10 days;
(4) dead chicken embryo step (4) obtained is isolated chorioallantoic membrane and allantoic fluid, in mass volume ratio 1g: the 10ml ratio joins by the mixture of chorioallantoic membrane and allantoic fluid the TNE damping fluid homogenate that PH is 7.4, by step (3) operation, obtain the supernatant liquor containing the Portunus trituberculatus Miers reovirus.
The TNE damping fluid compound method adopted in step (2) is: take 6.0578g Tris, and 12g Nacl, 1.8612g EDTA, add the 800ml distilled water to dissolve fully, with hydrochloric acid, regulates PH to 7.4, adds distilled water to be settled to 1000ml, autoclaving.
Compared with prior art, the invention has the advantages that: the present invention has proposed the chicken embryo culture method of Portunus trituberculatus Miers reovirus first, and the method has following advantage:
(1) mainly still rely on and collect morbidity after artificial challenge host crab or dead host crab obtains virus for the crab virus culture so far, not crab virus vitro culture correlative study report.The Portunus trituberculatus Miers reovirus chicken embryo culture related in the present invention is the vitro culture that the chicken embryo culture applies to crab virus first.Although the virus that the method for utilizing the virus host crab to carry out artificial culture can better obtain, exist the host crab with malicious risk.And the virus-free inapparent infection of SPF chicken embryo, the chicken embryo is very wide to viral sensitive range in addition, and multiple virus all can be suitable for;
(2) cultivate the Portunus trituberculatus Miers reovirus by the method for chicken embryo culture, overcome in Portunus trituberculatus Miers reovirus culturing process without the appropriate incubation system, the cell cultures cost is high, easily pollute and can not be gone down to posterity and cultivate and can't meet the shortcoming such as viral demand, and its virulence of virus that the present invention cultivates is suitable with primary virus virulence, occur to weaken, can be used for this virus continue go down to posterity and other any researchs for this virus.
(3) the crab viroid does not all have the method for cell cultures mostly, if so the crab viroid adopt cell cultures will be faced with such as culturing cell to select, the problems such as cell culture medium condition exploration.These processes often need to spend a large amount of energy and financial resources.Go down to posterity except above-mentioned band poison hidden danger, also have the problem that the laboratory animal purchase cost is high and use crab virus host crab to carry out virus, the factor such as cultivating condition and experiment management and seasonal climate also can affect to experiment in addition.The chicken embryo culture has well solved the problems referred to above.In the present invention, chicken embryo used is all purchased local special SPF kind chicken plant, obtain conveniently, cost is lower, the cost of having avoided exploitation cell cultures and purchase Portunus trituberculatus Miers to face is high, strict to requirement for experiment condition, the problem of the aspects such as aquaculture management, and can not be subject to the impact of seasonal climate, all can cultivate throughout the year.
(4) the chicken embryo of having inoculated the Portunus trituberculatus Miers reovirus is under above-mentioned corresponding culture temperature and incubation time, and virus can be bred preferably.
In sum, the chicken embryo culture method of Portunus trituberculatus Miers reovirus of the present invention provides a kind of more efficient, the extracorporeal culturing method of more applicable Portunus trituberculatus Miers reovirus, the Portunus trituberculatus Miers reovirus is carried out fast, effectively subculture in vitro separately is cultivated, overcome in Portunus trituberculatus Miers reovirus culturing process without the appropriate incubation system, existing cultural method is difficult to carry out the problem that virus goes down to posterity and cultivates, therefore the application of chicken embryo culture method in the Portunus trituberculatus Miers reovirus will be filled up the blank of aquatic animal virus chicken embryo culture system, and lay the foundation for later research and application.
Portunus trituberculatus Miers reovirus (Portunnus trituberculatus reovirus), strain is the R1015 strain, preserving number is CGMCC No.5379, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 21st, 2011, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The accompanying drawing explanation
Fig. 1 Portunus trituberculatus Miers reovirus chicken embryo culture electron microscopic observation result.
Embodiment
Below in conjunction with accompanying drawing, embodiment is described in further detail the present invention.
Following examples have been done to further illustrate to the present invention, but protection scope of the present invention is not limited to this, and protection domain is as the criterion with claim.
The chicken embryo culture method of Portunus trituberculatus Miers reovirus of the present invention, adopt the culture system in vitro of SPF chicken embryo as the Portunus trituberculatus Miers reovirus, select to have cultivated in advance the healthy chicken embryo of 3-4 days, previously prepared good Portunus trituberculatus Miers reovirus inoculation liquid is seeded on chick chorioallantoic membrane, continue to cultivate until the chicken embryo occurs dead, the dead chicken embryo that the collection incubation time is 3-10 days carries out the homogenate centrifugation and extracts the supernatant liquor containing the Portunus trituberculatus Miers reovirus, the strain of this Portunus trituberculatus Miers reovirus is the R1015 strain, preserving number is CGMCC No.5379, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 21st, 2011.Its concrete cultural method is as follows:
(1) choose the fresh kind of egg that meets the SPF specification, upwards 38 ℃ of hatchings of air chamber, relative humidity 50%, every 4 hours egg-turnings once, cultivate after 3-4 days, choose blood vessel thick and be the good chicken embryo of cherry healthy development at inside-paint discharge chamber edge, darkroom and indicate the great vessels present position, use in order to virus inoculation;
(2) choose some of Portunus trituberculatus Miers that infect reovirus, get the gill, muscle and internal organ, in mass volume ratio 1g: the 10ml ratio joins 12 ‰ the TNE damping fluid that PH is 7.4, homogenate in homogenizer by the mixture of the gill, muscle and internal organ, by homogenate in 4 ℃, 6000g, after centrifugal 20min, get containing viral supernatant liquor, the precipitation of centrifugal gained is resuspended with 12 ‰ TNE of 1～2ml, homogenate again, by homogenate obtains again homogenate in 4 ℃, 6000g, centrifugal 20min, the supernatant liquor that merges the two times centrifugal gained, first with the filter membrane suction filtration of 0.45 μ m, remove fragment of tissue by merging the gained supernatant liquor, use again the filter membrane suction filtration degerming of 0.22 μ m, after every milliliter of supernatant liquor after degerming adds 1000 unit penicillin and 1000ug Streptomycin sulphate, in the processing of spending the night of 4 ℃ of ice baths, obtain Portunus trituberculatus Miers reovirus inoculation liquid,
(3) choose health, the chicken embryo of well-developed 3-4 age in days, inoculum size by Portunus trituberculatus Miers reovirus inoculation liquid by 0.15-0.2ml is inoculated on the allantois of chicken embryo, cultivate 13-14 days by having inoculated viral chicken embryo under the constant temperature of 32.5 ℃, the dead chicken embryo that the collection incubation time is 3-10 days;
(4) dead chicken embryo step (4) obtained is isolated chorioallantoic membrane and allantoic fluid, in mass volume ratio 1g: the 10ml ratio joins by the mixture of chorioallantoic membrane and allantoic fluid 12 ‰ the TNE damping fluid homogenate that PH is 7.4, by step (3) operation, obtain the supernatant liquor containing the Portunus trituberculatus Miers reovirus;
(5) cultivation of going down to posterity: the method inoculated into chick embryo by the virus liquid of preparation in step (4) according to step (3).
In this specific embodiment, the middle kind of step (1) egg incubation time effect of inoculation the best after 3 days; The TNE damping fluid compound method adopted in step (2) is: take 6.0578g Tris, and 12g Nacl, 1.8612gEDTA, add the 800ml distilled water to dissolve fully, with hydrochloric acid, regulates PH to 7.4, adds distilled water to be settled to 1000ml, autoclaving.
In the present embodiment, dead chicken embryo is respectively organized the electron microscopic observation result as shown in fig. 1, sees a large amount of spherical viruses particles in each histocyte, in its form size and Portunus trituberculatus Miers tissue and after purifying the morphology of virus size basic identical.Be purified rear recurrence inoculation swimming crab and infect successfully, lethality rate 100%, clinical symptom is identical with the dead crab of natural occurrence, illustrates that virus virus virulence after the chicken embryo culture does not weaken.Virus is carried out the chicken embryo go down to posterity research in, having inoculated chicken embryo mortality ratio when cultivating end of separating the supernatant liquor containing the Portunus trituberculatus Miers reovirus obtained from dead chicken embryo tissue is 100%, control group (blank group, do not inoculate the chicken embryo of Portunus trituberculatus Miers reovirus) death do not appear in the chicken embryo, this has illustrated and has gone down to posterity viral chicken embryo to cultivate be feasible.Result shows to utilize chicken embryo culture Portunus trituberculatus Miers reovirus to achieve success.
Claims (3)
1. the chicken embryo culture method of a Portunus trituberculatus Miers reovirus, it is characterized in that: adopt the culture system in vitro of SPF chicken embryo as the Portunus trituberculatus Miers reovirus, select to have cultivated in advance the healthy chicken embryo of 3-4 days, previously prepared good Portunus trituberculatus Miers reovirus inoculation liquid is seeded on chick chorioallantoic membrane, under the constant temperature of 32.5 ℃, continue to cultivate until the chicken embryo occurs dead, the dead chicken embryo that the collection incubation time is 3-10 days carries out the homogenate centrifugation, obtain the supernatant liquor containing the Portunus trituberculatus Miers reovirus, the strain of described Portunus trituberculatus Miers reovirus is the R1015 strain, preserving number is CGMCC No.5379, be deposited in Chinese microbial preservation management committee's common micro-organisms center on October 21st, 2011.
2. the chicken embryo culture method of Portunus trituberculatus Miers reovirus according to claim 1 is characterized in that concrete operation steps is as follows:
(1) choose the fresh kind of egg that meets the SPF specification, upwards 38 ℃ of hatchings of air chamber, relative humidity 50%, every 4 hours egg-turnings once, cultivate after 3-4 days, choose blood vessel thick and be the good chicken embryo of cherry healthy development at inside-paint discharge chamber edge, darkroom and indicate the great vessels present position, use in order to virus inoculation;
(2) choose some of Portunus trituberculatus Miers that infect reovirus, get the gill, muscle and internal organ, in mass volume ratio 1g:10ml ratio, the mixture of the gill, muscle and internal organ is joined to the TNE damping fluid that PH is 7.4, homogenate in homogenizer, by homogenate in 4 ℃, 6000g, after centrifugal 20min, get containing viral supernatant liquor, the precipitation of centrifugal gained is resuspended with the TNE damping fluid of 1～2ml, homogenate again, by homogenate obtains again homogenate in 4 ℃, 6000g, centrifugal 20min, the supernatant liquor that merges the two times centrifugal gained, first with the filter membrane suction filtration of 0.45 μ m, remove fragment of tissue by merging the gained supernatant liquor, use again the filter membrane suction filtration degerming of 0.22 μ m, after every milliliter of supernatant liquor after degerming adds 1000 unit penicillin and 1000 μ g Streptomycin sulphates, in the processing of spending the night of 4 ℃ of ice baths, obtain Portunus trituberculatus Miers reovirus inoculation liquid,
(3) choose health, the chicken embryo of well-developed 3-4 age in days, inoculum size by Portunus trituberculatus Miers reovirus inoculation liquid by 0.15-0.2ml is inoculated on the allantois of chicken embryo, cultivate 13-14 days by having inoculated viral chicken embryo under the constant temperature of 32.5 ℃, the dead chicken embryo that the collection incubation time is 3-10 days;
(4) dead chicken embryo step (3) obtained is isolated chorioallantoic membrane and allantoic fluid, in mass volume ratio 1g:10ml ratio, the mixture of chorioallantoic membrane and allantoic fluid is joined to the TNE damping fluid homogenate that PH is 7.4, treatment process by homogenate in step (2) is operated homogenate, obtains the supernatant liquor containing the Portunus trituberculatus Miers reovirus.
3. the chicken embryo culture method of Portunus trituberculatus Miers reovirus according to claim 2, it is characterized in that: the TNE damping fluid compound method adopted in step (2) is: take 6.0578g Tris, 12g NaCl, 1.8612g EDTA, add the 800ml distilled water to dissolve fully, regulate PH to 7.4 with hydrochloric acid, add distilled water to be settled to 1000ml, autoclaving.
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| CN1940535A (en) * | 2006-09-27 | 2007-04-04 | 中山大学 | Ora serrata crab reovirus diagnostic reagent kit and its inspection |
| CN101098959A (en) * | 2004-10-22 | 2008-01-02 | 昂科利蒂克斯生物科技公司 | Improved viral purification methods |
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| CN101098959A (en) * | 2004-10-22 | 2008-01-02 | 昂科利蒂克斯生物科技公司 | Improved viral purification methods |
| CN1940535A (en) * | 2006-09-27 | 2007-04-04 | 中山大学 | Ora serrata crab reovirus diagnostic reagent kit and its inspection |
| CN102302772A (en) * | 2011-08-31 | 2012-01-04 | 齐鲁动物保健品有限公司 | Duck hemorrhagic ovaritis (DHO) inactivated vaccine and preparation method thereof |
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| Guneratne J.等."Some observations on the isolation and cultivation of avian reoviruses".《Avian Pathology》.1982,第11卷(第3期),第453－462页. |
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