Method for constructing muscovy duck reovirus (MDRV) reverse genetic system

A technology of reovirus and reverse genetics, which is applied in the field of building Muscovy duck reovirus reverse genetic system, can solve problems such as stagnant research, lack of technical means, and inability to carry out MDRV genome function research, achieving purity High, toxic effect

Active Publication Date: 2019-02-12
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

[0005] Due to the lack of technical means, it is impossible to carry out the research on the function of MDRV genome, which leads to the stagnation of related research

Method used

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  • Method for constructing muscovy duck reovirus (MDRV) reverse genetic system
  • Method for constructing muscovy duck reovirus (MDRV) reverse genetic system
  • Method for constructing muscovy duck reovirus (MDRV) reverse genetic system

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Embodiment Construction

[0032] The following examples further describe the present invention.

[0033] Step 1, virus isolates:

[0034]MDRV Fujian representative strain MW9710 was isolated, identified and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0035] Step 2, viral RNA extraction:

[0036] MDRV genomic RNA is prepared by TRIzol reagent, and the specific operation steps are as follows:

[0037] a. Collect 250 μL of MDRV virus culture solution and transfer it to a 1.5 mL centrifuge tube.

[0038] b. Add 750 μL TRIzol, vortex to mix evenly, and let stand for 5 min.

[0039] c. Add 150 μL CHCl3, vortex to mix evenly, let stand for 5 min

[0040] d. Centrifuge at 12,000×g for 10 min. Transfer the supernatant to a new 2 mL centrifuge tube, add 500 μL isopropanol, invert up and down 6-8 times, and mix well.

[0041] e. Centrifuge at 12,000×g for 10 min. Discard the supernatant, add 1mL absolute ethanol, and vortex to mix well....

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Abstract

The invention discloses a method for constructing a muscovy duck reovirus (MDRV) reverse genetic system. Viral genome RNA is extracted from cell culture poison containing the muscovy duck reovirus; adesigned specific primer and the viral genome RNA serving as a template are utilized to perform RT-PCR amplification on gene segments of an MDRV complete sequence, enzyme digestion is performed on target gene segments to connect the target gene segments into 10 gene segments of the MDRV, the 10 gene segments are connected with a carrier, and positive bacterial colonies are picked to perform sequencing and verification; positive plasmids of the 10 segments of the MDRV are mixed according to the proportion to perform cotransfection on single-layer 293T cells. After passage is performed on the cells for 15th generation, cytopathy becomes stable, and MDRV recombinant virus can be obtained.

Description

technical field [0001] The invention relates to a method for constructing a Muscovy duck reovirus (MDRV) reverse genetic system. Background technique [0002] Muscovy duck reovirus disease is caused by muscovy duck reovirus (MDRV). The diseased duck is characterized by soft feet, a large number of gray-white necrosis spots on the surface of the liver and spleen, and kidney enlargement and hemorrhage. Infectious diseases, and the immune organs are severely damaged, showing immunosuppression, and may play the role of virus carrier and virus storage host in the process of waterfowl influenza transmission and epidemic. Therefore, this disease has become one of the main diseases that seriously threaten the waterfowl breeding industry at present. [0003] According to the report of the Seventh International Committee on Taxonomy of Viruses (2000, Van Regenmortal et al.), Muscovy duck reovirus is a member of subgroup II avian orthoreovirus of the family Reoviridae. Under the elec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/85C12N5/10
CPCC12N15/85C07K14/005C12N2720/12222
Inventor 林锋强陈少莺王劭
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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