Method for constructing reverse genetic system of red-spotted grouper nervous necrosis virus

A red-spotted grouper, nerve necrosis technology, applied in the field of aquatic animal virus genetic engineering, can solve problems such as low transfection efficiency, and achieve the effect of convenient virus rescue

Active Publication Date: 2020-07-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2001, Japanese scholar Naoki successfully established the reverse genetic operating system of RGNNV using the T7 polymerase promoter, but there is no report on the establishment of the reverse genetic operating system of RGNNV using the CMV promoter.
However, the low transfection efficiency of many fish cells is a limiting factor in virus rescue

Method used

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  • Method for constructing reverse genetic system of red-spotted grouper nervous necrosis virus
  • Method for constructing reverse genetic system of red-spotted grouper nervous necrosis virus
  • Method for constructing reverse genetic system of red-spotted grouper nervous necrosis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 compares the replication effect of RGNNV in SSN-1 cells and GF-1 cells

[0025] GF-1 and SSN-1 cells were infected with RGNNV, and a control group (Mock) not infected with RGNNV was set at the same time. Cell pathological changes were observed and photographed 72 hours after infection. Discard the culture medium, wash twice with pre-cooled PBS, then add an appropriate amount of pre-cooled PBS to incubate the cells for 2-3 minutes, gently blow the cells from the culture plate with a pipette gun, and collect them into a 1.5ml centrifuge tube. Centrifuge at 3,000 g for 10 min at 4°C. Pour off the supernatant, resuspend the cell pellet with 40-100 μl of pre-cooled PBS, add the corresponding volume of 5×SDS-PAGE loading buffer, and cook in boiling water for 10 minutes.

[0026] Clamp the glass plate for pouring the polyacrylamide gel, configure the separating gel according to the instructions, pour about 5ml of the prepared separating gel into the gap between the ...

Embodiment 2

[0029] The construction of embodiment 2 RGNNV genome plasmid

[0030]The synthetic HamRz-BamHI-HdvRz fragment (with HindIII and XbaI restriction sites at both ends) was digested by HindIII and XbaI (purchased from Treasure Bioengineering (Dalian) Co., Ltd.), while pcDNA3.1(+) Plasmid ( figure 2 ) by HindIII and XbaI digestion, the digested fragment and pcDNA3.1 (+) plasmid were recovered, and connected with T4 DNA ligase (purchased from Treasure Bioengineering (Dalian) Co., Ltd.), the connection system was as follows:

[0031]

[0032] Water bath at 16°C overnight, transform DH5α-competent bacteria, culture at 37°C for 12-16 hours, pick bacteria for PCR identification of bacteria liquid, and send positive clones for sequencing (Wuhan Qingke Biotechnology Co., Ltd.), and the obtained recombinant plasmid was named pcDNA-Ham -BamHI-Hdv.

[0033] Table 1 Primer Sequence

[0034]

[0035] Infect SSN-1 cells with RGNNV, 24 hours after infection, discard the culture medium,...

Embodiment 3

[0045] Embodiment 3SSN-1 cell compares with 293T cell transfection efficiency

[0046] Prepare the SSN-1 cells and 293T cells for transfection 24 hours before transfection. Dilute 4 μg of the green fluorescent plasmid PEGFP-N1 into 400 μl of serum-free OPTI-MEM medium, add 12 μl of transfection reagent TransIntroTM ELTransfection Reagent (Beijing Quanshijin Biotechnology Co., Ltd.), blow gently for more than 10 times, and mix well , combined at room temperature for 30min. Wash 2 times with serum-free and antibiotic-free OPTI-MEM for SSN-1 cells or 293T cells that have been cultured for about 18-24 hours in a 6-well culture plate with 80%-90% abundance, uniform distribution, and good growth conditions. 200 μl of the mixture of plasmid and transfection reagent was added to each cell, and 800 μl of OPTI-MEM was added to cover the cells. SSN-1 cells in a 25 °C incubator, 293T cells at 37 °C, 5% CO 2 in the incubator. After adsorption for 6-8 hours, replace with 2 ml of MEM (SS...

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Abstract

The invention belongs to the technical field of virus gene engineering of aquatic animals, and particularly relates to a method for constructing a reverse genetic system of red-spotted grouper nervousnecrosis virus (RGNNV). According to the invention, CMV is used as a promoter to construct an eukaryotic expression plasmid containing an RGNNV genome; the RGNNV is rescued by adopting a method of combining human embryo kidney 293T cells with high transfection efficiency and striped snakehead cells (SSN-1) which efficiently replicate the RGNNV, so that the problem of low transfection efficiency of fish cells is solved.

Description

technical field [0001] The invention belongs to the technical field of aquatic animal virus genetic engineering, and specifically relates to a method for constructing a reverse genetic system of red grouper neuronecrosis virus. Background technique [0002] Nervous necrosis virus (NNV) is a member of the Nodaviridae family. The disease caused by its infection is called viral nerve necrosis (viral nervous necrosis, VNN) or viral encephalopathy and retinopathy (viral encephalopathy and retinopathy, VER). It has now been reported in more than 120 different farmed and wild fish species, causing huge economic losses to the aquaculture industry. In 2001, Lin Li et al. isolated the virus from naturally occurring red-spotted grouper fry in Daya Bay, Guangdong Province, China, and it was red-spotted grouper nervous necrosis virus (RGNNV). RGNNV is a positive-strand RNA virus, and the viral genome contains two segments of RNA (RNA1 and RNA2). RNA1 encodes viral RNA polymerase, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N7/00C12R1/93
CPCC12N15/85C12N7/00C12N2800/107C12N2800/106C12N2830/60C12N2770/30052
Inventor 涂加钢张永安
Owner HUAZHONG AGRI UNIV
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