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
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Examples
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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Description
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Application Information
- IPC
- C12N15/113; C12N15/867; A61K48/00; A61K31/7105; A61P31/14
- CPC
- C12N15/1131; C12N15/86; A61K31/7105; A61P31/14; C12N2310/14; C12N2310/531; C12N2740/15043; C12N2800/106
- Inventors
- 袁雪梅; 吕孙建