CN102613112B - A kind of method for building up of prawn ' s virus infection model - Google Patents
A kind of method for building up of prawn ' s virus infection model Download PDFInfo
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- CN102613112B CN102613112B CN201210062792.6A CN201210062792A CN102613112B CN 102613112 B CN102613112 B CN 102613112B CN 201210062792 A CN201210062792 A CN 201210062792A CN 102613112 B CN102613112 B CN 102613112B
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- prawn
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The present invention relates to a kind of method for building up of prawn ' s virus infection model, comprising: the preparation of (1) WSSV virus purification and conserving liquid; (2) variable concentrations WSSV conserving liquid infects Environment of Litopenaeus vannamei Low; (3) prawn cumulative mortality observation determines the prawn ' s virus infective dose of this RNA the Study of Interference.By the prawn ' s virus infection model favorable reproducibility of method establishment of the present invention, RNA the Study of Interference credible result degree is high; Easy and simple to handle, criterion is distinct, is not only applicable to the antiviral study of RNA interference, also can be used for RNA interference preparation applying in aquatic products practice in the future.
Description
Technical field
The invention belongs to antiviral study field, particularly a kind of method for building up of prawn ' s virus infection model.
Background technology
The features such as prawn fertility is strong, and growth is rapid, and meat flavour is delicious.And Environment of Litopenaeus vannamei Low has the stronger feature of halophile and premunition widely, thus by world's cultivating large area.In China's cultured prawn kind, more than 80% is Environment of Litopenaeus vannamei Low.
Shrimp white spot syndrome virus (whitespotsyndromevirus, WSSV) is with its pantropic to host, highly pathogenic and high lethality rate and occupy first of prawn ' s virus.Generally in 3 ~ 10d, lethality can up to 100% to infect prawn.Because prawn is the same with other invertebrates, the shortage of specific immune mechanisms causes its virus disease control to put prevention first with non-specific always, lacks specific prevention and controls.Therefore, WSSV almost every average annual China's shrimp culture industry of giving brings tremendous economic to lose, and the phenomenon that pool mouth causes prawn to be had no harvest because breaking out hickie disease also often has generation.In view of the critical role of China's Environment of Litopenaeus vannamei Low in China's culture fishery, the method for seeking efficient, special control WSSV becomes the key guaranteeing that China's shrimp culture industry develops in a healthy way.
RNA disturbs (RNAi) technology to be the specific RNA degradation process of being induced by double-stranded RNA (dsRNA) in eukaryotic, in cellular level and animal and plant body, all show efficiently special anti-virus ability.Correlative study in recent years proves, RNAi also can excite efficient antiviral effect in aquatic invertebrate body.Due to WSSV genome sequence clearly, therefore, use RNA perturbation technique anti-WSSV, Effect of Anti virus special preparation to be completely possible.
But we RNA in vertebrate disturbs antiviral study method and is not suitable for the such aquatic invertebrate of prawn.Because the continuous cell line that prawn is unstable, relevant RNA the Study of Interference can only directly in prawn body level carry out; And the prawn of different batches due to individual difference very large, they must be different to the neurological susceptibility of virus.Carrying out RNA in such a situa-tion disturbs antivirus test will certainly affect reappearance and the confidence level of experimental result.Therefore, set up a metastable RNA the Study of Interference system RNA the Study of Interference is carried out for level in prawn body to be absolutely necessary.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method for building up of prawn ' s virus infection model, the prawn ' s virus infection model favorable reproducibility that the method is set up, and RNA the Study of Interference credible result degree is high.
The method for building up of a kind of prawn ' s virus infection model of the present invention, comprising:
(1) Environment of Litopenaeus vannamei Low of artificial infection WSSV sequela death removed appendage, carapace and hepatopancrease and weigh, press mass volume ratio 1: 10 with 0.01MPBS to dilute, glass homogenizer homogenate, homogenate is in 4 DEG C of centrifugal 20min, supernatant successively with filter paper and 0.45 μm of membrane filtration degerming and be distributed into viral conserving liquid ,-80 DEG C are frozen for subsequent use;
(2) body weight 7 ~ 9g, PCR detect negative healthy Environment of Litopenaeus vannamei Low after indoor pond supports 1 week temporarily, and random packet is often organized 14, supported temporarily respectively in glass bucket; Before each RNA interference experiment, viral conserving liquid is diluted to 10
-(n-2), 10
-(n-1), 10
-n, 10
-(n+1), 10
-(n+2)the infection liquid of totally 5 concentration, with insulin syringe respectively at Environment of Litopenaeus vannamei Low the 4th uromere place intramuscular injection, every tail 50 μ L, control group injects 0.01MPBS with method; Prawn culturing water temperature controls at 27 ~ 28 DEG C, changes water 1/2,24h every day and continues inflation; Observe four every day, pick dead and dying prawn, record death toll, continuous observation 8d;
(3), according to criterion, when carrying out RNA interference experiment, virus infections liquid concentration is that viral conserving liquid does 10
-ndoubly dilution, determines the virus infections amount of prawn afterwards, sets up virus infections model.
In described step (2), the value of n is, after the viral conserving liquid of new collection infects prawn in advance, cause the greatest dilution of more than 90% prawn death in 7 days.
Often criticize prawn Modling model virus in described step (2) all to collect and the viral conserving liquid of packing with criticizing from (1).
Observation number of days in described step (2) is 8 days.
Criterion in described step (3) is that the cumulative mortality infecting prawn in 7d is greater than the dilution factor of the maximum viral conserving liquid of the group of 90% as RNA interference virus infections concentration.
Beneficial effect
By the prawn ' s virus infection model favorable reproducibility of method establishment of the present invention, RNA the Study of Interference credible result degree is high; Easy and simple to handle, criterion is distinct, is not only applicable to the antiviral study of RNA interference, also can be used for RNA interference preparation applying in aquatic products practice in the future.
Accompanying drawing explanation
Fig. 1 is the foundation of Environment of Litopenaeus vannamei Low WSSV infection model; What show in figure is the cumulative mortality that Environment of Litopenaeus vannamei Low infects after different dilution WSSV conserving liquid in 7 days; In figure, E-1 to E-5 represents the dilution factor of the WSSV conserving liquid of different group respectively: 10
-1to 10
-5;
Fig. 2 is the PCR testing result of WSSV in different shRNA processed group (comprising the positive and negative control group) Environment of Litopenaeus vannamei Low body; Wherein, A1 and A2 is the result of first time experiment; A1 is the WSSV median infective dose of each group of shrimp samples; A2 is with 18S rDNA average expression amount in group prawn body; B1 and B2 is for repeating the result of testing (another batch of prawn ' s virus infection model) for the second time.In figure, 2 ~ 6 row represent the result that different RNA disturbs processed group respectively; 1 and 7 represent result that is positive and negative control respectively.
Embodiment
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.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
The preparation of WSSV virus purification and viral conserving liquid
The Environment of Litopenaeus vannamei Low of artificial infection WSSV sequela death is removed appendage, carapace and hepatopancrease and weighs, dilute with 0.01MPBS (pH7.4) 1: 10 (g/mL), glass homogenizer homogenate, homogenate is in 4 DEG C of centrifugal 20min of 4000rpm, supernatant successively with filter paper and 0.45 μm of membrane filtration degerming and be distributed into viral conserving liquid ,-80 DEG C are frozen for subsequent use.
Variable concentrations WSSV conserving liquid infects Environment of Litopenaeus vannamei Low
What body weight 7 ~ 9g, PCR detection was negative newly purchases healthy Environment of Litopenaeus vannamei Low after indoor pond supports 1 week temporarily, and random packet is often organized 14, supported respectively temporarily in the white glass bucket of 90L.Viral conserving liquid is diluted to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5the infection liquid (knowing dilution range after infecting prawn in advance by new isolated viral conserving liquid) of totally 5 concentration, with insulin syringe respectively at Environment of Litopenaeus vannamei Low the 4th uromere place intramuscular injection, every tail 50 μ L, control group injects 0.01MPBS (pH7.4) with method.Prawn culturing water temperature controls at 27 ~ 28 DEG C, changes water 1/2,24h every day and continues inflation.Observe four every day, pick dead and dying prawn in time, record death toll, continuous observation 8d.
Prawn cumulative mortality observation determines the prawn ' s virus infective dose of this RNA the Study of Interference
As shown in Figure 1, along with WSSV infects the reduction of concentration, the time of occurrence of Environment of Litopenaeus vannamei Low peak mortality is delayed relatively, and in observing time, prawn accumulation death toll reduces relatively; When the dilution factor of WSSV conserving liquid is 10
-1~ 10
-3times time, in 7d, prawn is almost all dead (being greater than 90%), and dilution factor is 10
-4times time death toll obviously reduce (being less than 90%).Cumulative mortality according to infecting prawn in criterion of the present invention: 7d is greater than the maximum viral conserving liquid dilution factor of the group of 90% as RNA interference virus infections concentration.Therefore, when this batch of prawn carries out RNA interference experiment, applicable virus infections liquid concentration is that viral conserving liquid does 10
-3doubly dilution (n=3).Virus infections volume due to every prawn is fixing (50 μ L/ only), and thus the virus infections amount of prawn is also determined thereupon, and virus infections model is successfully set up.
Above method establishment WSSV infection model is utilized to carry out the research that RNA disturbs anti-WSSV, experiment repetition 2 times.As shown in Figure 2, the testing result of semiquantitive PCR to prawn body inner virus infective dose is basically identical afterwards in twice experiment (in corresponding diagram A, B).And experiment also shows, infective dose result and the prawn lethality experimental result of WSSV are also basically identical.
Claims (2)
1. a method for building up for prawn ' s virus infection model, comprising:
(1) Environment of Litopenaeus vannamei Low of artificial infection WSSV sequela death removed appendage, carapace and hepatopancrease and weigh, press mass volume ratio 1:10 with 0.01MPBS to dilute, glass homogenizer homogenate, homogenate is in 4 DEG C of centrifugal 20min, supernatant successively with filter paper and 0.45 μm of membrane filtration degerming and be distributed into viral conserving liquid ,-80 DEG C are frozen for subsequent use;
(2) body weight 7 ~ 9g, PCR detect negative healthy Environment of Litopenaeus vannamei Low after indoor pond supports 1 week temporarily, and random packet is often organized 14, supported temporarily respectively in glass bucket; Before each RNA interference experiment, viral conserving liquid is diluted to 10
-(n-2), 10
-(n-1), 10
-n, 10
-(n+1), 10
-(n+2)the infection liquid of totally 5 concentration, with insulin syringe respectively at Environment of Litopenaeus vannamei Low the 4th uromere place intramuscular injection, every tail 50 μ L, control group injects 0.01MPBS with method; Prawn culturing water temperature controls at 27 ~ 28 DEG C, changes water 1/2,24h every day and continues inflation; Observe four every day, pick dead and dying prawn, record death toll, continuous observation 8d; Wherein, the value of n is, after the viral conserving liquid of new collection infects prawn in advance, cause the greatest dilution of more than 90% prawn death in 7 days;
(3), according to criterion, when carrying out RNA interference experiment, virus infections liquid concentration is that viral conserving liquid does 10
-ndoubly dilution, determines the virus infections amount of prawn afterwards, sets up virus infections model; Wherein, criterion is that the cumulative mortality infecting prawn in 7d is greater than the dilution factor of the maximum viral conserving liquid of the group of 90% as RNA interference virus infections concentration.
2. the method for building up of a kind of prawn ' s virus infection model according to claim 1, is characterized in that: often criticize prawn Modling model virus in described step (2) and all collect and the viral conserving liquid of packing with criticizing from (1).
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| CN102978171A (en) * | 2012-12-07 | 2013-03-20 | 广西壮族自治区水产研究所 | Method for establishing prawn virus isolation, identification and infection model |
| CN104278072A (en) * | 2013-07-05 | 2015-01-14 | 徐州工程学院 | Method for bacteriostatic effect evaluation by inoculation of Vibrio parahaemolyticus in shrimps |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1926970A (en) * | 2006-09-26 | 2007-03-14 | 广东恒兴集团有限公司 | Process for selecting and breeding litopenaeus vannamei parents with white spot syndrome virus resisting character |
| CN102007882A (en) * | 2010-12-17 | 2011-04-13 | 惠东县巽寮镇瑞帝养殖场 | Method for breeding high-quality penaeus vannamei boone parent shrimps |
| CN102106291A (en) * | 2011-01-31 | 2011-06-29 | 中国水产科学研究院东海水产研究所 | Specific-pathogen-free litopenaeus vannamei seedlings breeding method for southern China |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1926970A (en) * | 2006-09-26 | 2007-03-14 | 广东恒兴集团有限公司 | Process for selecting and breeding litopenaeus vannamei parents with white spot syndrome virus resisting character |
| CN102007882A (en) * | 2010-12-17 | 2011-04-13 | 惠东县巽寮镇瑞帝养殖场 | Method for breeding high-quality penaeus vannamei boone parent shrimps |
| CN102106291A (en) * | 2011-01-31 | 2011-06-29 | 中国水产科学研究院东海水产研究所 | Specific-pathogen-free litopenaeus vannamei seedlings breeding method for southern China |
Non-Patent Citations (1)
| Title |
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| 对虾白斑综合征病毒在螯虾动物模型的感染特性;朱建中等;《水产学报》;20010228;第25卷(第1期);第47-49页 * |
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