ShRNA for inhibiting replication of micropterus salmoides rhabdovirus and application of shRNA

The technology of a lentiviral vector and a recombinant vector is applied to shRNA for inhibiting the replication of largemouth rhabdovirus G glycoprotein and its application field, which can solve the problems of economic loss of largemouth bass and the incidence of largemouth bass, and achieve inhibition of glycoprotein The effect of copying, broad application prospects

Active Publication Date: 2021-03-30
ZHEJIANG INST OF FRESH WATER FISHERIES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the disease is severe, it will cause 80% of the largemouth bass in the breeding pond to be sick, and the mortality rate can reach 90% in a short period of time, and it is ineffective with antibacterial drugs
The disease is also typically contagious, causing huge economic losses to largemouth bass farming in my country, for example.

Method used

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  • ShRNA for inhibiting replication of micropterus salmoides rhabdovirus and application of shRNA
  • ShRNA for inhibiting replication of micropterus salmoides rhabdovirus and application of shRNA
  • ShRNA for inhibiting replication of micropterus salmoides rhabdovirus and application of shRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening of Largemouth Bass Rhabdovirus Interference Targets

[0033] Aiming at the G, M, N, L gene sequences of largemouth bass rhabdovirus (GenBank: MK397811.2), 12 siRNA molecules were designed and synthesized (see Table 1). The 12 siRNA molecules were transfected into grass carp ovary cells (CO) by lipofection method, and the transfection steps were as follows: 3000 (Thermo Scientific company product) manual, 6h later with 100 TCID 50 The MSRV infected CO cells, and the cell virus liquid was collected 72 hours after the completion of the virus inoculation, and the total RNA was extracted by Trizol reagent (Takara Company) and reverse-transcribed into cDNA, and then Real-time PCR was performed. The specific method refers to the literature (Yuan Xuemei. Dakou Isolation and culture of black bass rhabdovirus and preparation of yolk antibody [J]. Fishery Science Advances, 2020, 41(3):151-157), using qPCR Mix (product of Dongyangfang (Shanghai) Biotechnolog...

Embodiment 2

[0039] The construction of embodiment 2 shRNA expression vector

[0040] 1. For the MSRV interference target screened in Example 1, design and synthesize shRNA expression vectors, design route:

[0041] TTCAAGAGA was selected as the stem-loop structure in the shRNA template to avoid the formation of a termination signal, and the transcription termination sequence of the shRNA adopted the T6 structure. CACC was added to the 5' end of the sense strand template, which was complementary to the sticky end formed after BbsI digestion; AGCT was added to the 5' end of the antisense strand template, which was complementary to the sticky end formed after HindIII digestion.

[0042] 2. The target sequence design of the target gene is shown in Table 3 below:

[0043] table 3

[0044] sequence name Sence (as shown in SEQ ID NO.31) Antience (as shown in SEQ ID NO.32) sh384-MSRVG-874 GCACAAATCGCCTGCATAT ATATGCAGGCGATTTGTGC

[0045] (1) sh384-MSRV G-874

[0046] S...

Embodiment 3

[0067] Embodiment 3 shRNA expression vector antiviral effect in vitro

[0068] The eukaryotic expression vector pSGU6 / GFP / Neo-G-874 was constructed for the interference target SiG874, the recombinant plasmid was extracted using a kit (promega company product), and transfected into CO cells, and then the transfected cells were challenged. The cell virus liquid was collected at different times, and Real-time PCR (same as Example 1) was used to detect the expression of virus mRNA, and according to the routine laboratory method (Xu Yang. Isolation and identification of two strains of grass carp reovirus Jiangxi strain[J]. Freshwater Fisheries, 2010(03): 44-49) Detection of virus TCID 50 , Westerin-blot detection of viral protein expression, and MTT method (Yuan Xuemei. Isolation and cultivation of rhabdovirus from largemouth bass and preparation of yolk antibody [J]. Advances in Fishery Science, 2020,41(3):151-157) Cell survival was detected at 72 h after virus infection. The re...

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Abstract

The invention discloses shRNA for inhibiting the replication of micropterus salmoides rhabdovirus and an application of the shRNA, and belongs to the technical field of biological detection. The nucleotide sequence of the shRNA provided by the invention is as shown in SEQ ID No.35. The invention also provides a recombinant vector containing a DNA fragment for encoding the shRNA, a strain containing the recombinant vector, and applications of the recombinant vector and the strain in preparation of drugs for inhibiting the replication of micropterus salmoides rhabdovirus G glycoprotein. The shRNA recombinant vector provided by the invention is safe and non-toxic, can significantly inhibit the replication of the micropterus salmoides rhabdovirus G glycoprotein, and has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an shRNA for inhibiting the replication of largemouth bass rhabdovirus and its application, in particular to an shRNA for inhibiting the replication of largemouth bass rhabdovirus G glycoprotein and its application. Background technique [0002] Largemouth bass (Micropterus salmoides) is commonly known as California bass, black bass. Belonging to Perciformes Perciforings, Perciformes Porcoidei, Sunfish family Cehtrachidae, black perch Micropterus, native to the Mississippi River system in California, USA, is a delicious meat, strong disease resistance, fast growth, easy to catch Valuable carnivorous fish. Largemouth bass have a wide temperature range and strong resistance to low oxygen. They like to inhabit sandy or sandy and muddy still water without turbidity. [0003] In recent years, largemouth bass has been continuously threatened by diseases caused by various...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867A61K48/00A61K31/7105A61P31/14
CPCC12N15/1131C12N15/86A61K31/7105A61P31/14C12N2310/14C12N2310/531C12N2740/15043C12N2800/106
Inventor 袁雪梅吕孙建张海琪刘莉杭小英施伟达于喆
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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