RNA Strand Displacement Assay for Hepatitis E Virus Helicase
戊型肝炎病毒解旋酶的RNA链置换测定
发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2198 浏览次数: 7996
评审: Yannick DebingVamseedhar RayaproluDavid Paul
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参见作者原研究论文
The authors used this protocol in:
Apr 2016
Abstract
The hepatitis E virus (HEV) helicase uses ATP to unwind the RNA duplexes. This is an essential step for viral replication. This protocol aims to measure the double strand RNA unwinding activity of the HEV helicase.
Keywords: Hepatitis E virus (戊型肝炎病毒)
RNA virus (RNA病毒)
Helicase (解旋酶)
Helicase assay (解旋酶测定)
Viral replication (病毒复制)
Nonradioactive helicase assay (非放射性解旋酶测定)
Background
The RNA unwinding activity of HEV helicase has been measured using radiolabeled double stranded RNA (dsRNA, Karpe et al., 2010). We have established a non-radioactive assay protocol for measuring the dsRNA unwinding activity of the HEV helicase. This assay utilizes a fluorescently tagged RNA to measure the activity of the HEV helicase protein purified from human hepatoma cell, thus eliminating the need to handle radioactive material.
Materials and Reagents
- 1.5 ml microcentrifuge tube
- 60 mm plates
- 1.6 ml RNase free microcentrifuge tube
- PVDF (Polyvinylidene fluoride) membrane (Pall, catalog number: BSP0149 )
- Mammalian expression plasmids (details in Nair et al., 2016):
- pCDNA5 Helicase-flag [HEV helicase sequence was PCR amplified using primers: pCDNA5 HEL-flag FP, pCDNA5 HEL-flag RP; digested with HindIII and ligated into pCDNA5 vector digested with HindIII and EcoRV]
- pUNO ORF2-flag [ORF2 with C-terminal Flag tag was PCR amplified with primers: pUNO ORF2-flag FP, pUNO ORF2-flag RP; digested with BglII and ligated into pUNO vector digested with NheI [blunted] and BamHI]
- Huh7 human hepatoma cells (obtained from Dr. C. M. Rice; Blight et al., 2000)
- RNA oligos: HRNA1 5’-UUUUUUUUUUUUCGCUGAUGUCGCCUGG-3’ and HRNA2 5’-[6FAM] CCAGGCGACAUCAGCG-3’ (Sigma-Aldrich, USA)
- Nuclease-free water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9937 )
- Ethidium bromide solution (Sigma-Aldrich, catalog number: E1510 )
- Glycogen (Sigma-Aldrich, catalog number: G0885 )
- Ethanol (EMD Millipore, catalog number: 100983 )
- Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
- DMEM
- 10% fetal bovine serum (FBS)
- Phosphate-buffered saline (PBS) (Bio Basic, catalog number: PD0100 )
- Flag M2 agarose resin (Sigma-Aldrich, catalog number: A2220 )
- Flag peptide (Sigma-Aldrich, catalog number: F4799 )
- DEPC-treated water (Thermo Fisher Scientific, AmbionTM, catalog number: AM9922 )
- Octa-probe antibody (Santa Cruz Biotechnology, catalog number: sc-807 )
- Skimmed milk (Sigma-Aldrich, catalog number: 70166 )
Note: This product has been discontinued. - Anti-rabbit IgG Horseradish peroxidase(HRPO) (Santa Cruz Biotechnology, catalog number: sc-2004 )
- Clarity Western ECL blotting substrate (Bio-Rad Laboratories, catalog number: 1705061 )
- Silver stain kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 24612 )
- Bradford assay reagent (Bio-Rad Laboratories, catalog number: 5000002 )
- RNAsin
- ATP
- Proteinase K (Sigma-Aldrich, catalog number: P2308 )
- Glycogen (Sigma-Aldrich, catalog number: G0885 )
- Tris (Sigma-Aldrich, catalog number: T6066 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
- Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
- SDS (Sigma-Aldrich, catalog number: L3771 )
- Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’,-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: 03777 )
- Triton X-100 (Sigma-Aldrich, catalog number: 93418 )
Note: This product has been discontinued. - Sodium pyrophosphate tetrabasic decahydrate (Na4P2O4·10H2O) (Sigma-Aldrich, catalog number: S6422 )
- β-glycerol phosphate (Sigma-Aldrich, catalog number: 50020 )
- Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: S6508 )
- Protease inhibitor cocktail tablet (Roche Diagnostics, catalog number: 04693132001 )
- HEPES (Sigma-Aldrich, catalog number: H8651 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
- BSA
- DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
- Tween 20
- Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
- Xylene cyanol FF (Sigma-Aldrich, catalog number: X4126 )
- Annealing buffer (see Recipes)
- Soaking buffer (see Recipes)
- IP buffer (see Recipes)
- Strand displacement buffer (see Recipes)
- 5x termination buffer (see Recipes)
- 2x proteinase K buffer (see Recipes)
- Phosphate buffered saline with 0.1% Tween 20 (PBST) (see Recipes)
- 50 mg/ml proteinase K (see Recipes)
- 10 mg/ml glycogen (see Recipes)
- RNA loading dye (see Recipes)
Equipment
- Vertical electrophoresis mini apparatus
- Gel documentation system (Bio-Rad Laboratories, model: ChemiDocTM MP )
- NanoDrop (Thermo Fischer Scientific, USA)
- Heat block
- Water bath
- Refrigerated table top centrifuge
- -80 °C deep freezer
- Flip flop rocker
- 5% CO2 incubator
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Nair, V. P. and Surjit, M. (2017). RNA Strand Displacement Assay for Hepatitis E Virus Helicase. Bio-protocol 7(7): e2198. DOI: 10.21769/BioProtoc.2198.
- Nair, V. P., Anang, S., Subramani, C., Madhvi, A., Bakshi, K., Srivastava, A., Shalimar, Nayak, B., Ct, R. K. and Surjit, M. (2016). Endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype-1 hepatitis E virus. PLoS Pathog 12(4): e1005521.
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分类
微生物学 > 微生物生物化学 > RNA
生物化学 > RNA > RNA-蛋白质相互作用
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