Ucts were then analyzed by 1.3 agarose-formaldehyde gel electrophoresis and RNA quantity was measured by imageJ system. For study the degradation pattern, and also to rule out residual endogenous RNase activity from E. coli, vhs translated from RRL was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14960617 also used for RNA assay following the method described previously [8]. Briefly, RNA substrates were produced and labeled with -[p32] ATP by in vitro transcription and then incubated with assay 1-phenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole buffer (mock control), RRL reagent (negative control), or RRL translated vhs at 37 for the indicated time points (0, 15, 30 min). RNA products were recovered byLiu et al. Vet Res (2015) 46:Page 7 ofRNeasy?mini kit (QIAGEN, Center Mainz, Germany) and analyzed by 1.3 formaldehyde agarose gel electrophoresis and autoradiography image.Northern blot analysisstatistically significant difference and was shown asterisk sign (*) in all figures.ResultsIn vitro assay of ribonuclease activity mediated by PrV vhsAccumulation of reporter gene RNA was detected by Northern blot analysis using probe with sequences complementary to R-luc. Briefly, R-luc probes were generated by PCR with primer set (Rluc-F: TCCGCTAGAGCC ACCATGAC and Rluc-R: GGCCCTTCACCTTCACG AAC). The PCR amplification conditions were 95 for 5 min followed by 30 cycles of denaturation at 95 for 1 min, annealing at 55 for 2 min, extension at 72 for 2 min, and a final extension at 72 for 7 min. Radiolabeled DNA probe was generated by random primer labeling with -[p32] dATP. Total RNA was harvested from cells co-transfected with reporter plasmid pRluc and plasmid expressing wild type PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9144744 (WT) vhs (HSV-1, or PrV), or PrV vhs mutants with deletion of individual boxes by RNeasy?mini kit (QIAGEN). Subsequently, total RNA was separated on a 1.3 denaturing formaldehyde gel and transferred to a Hybond-N+ membrane (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA). Following UV-crosslinking fixation and pre-hybridization (for 1 h at 68 in prehybridization buffer 0.5 M sodium phosphate, 7 SDS and bmjgh-2016-000132 1 mM EDTA), the membrane was hybridized with radiolabeled DNA probe at 68 overnight. After washing steps, the membrane was exposed to a phosphoimage screen (Fuji, Tokyo, Japan) and detected with a Bio-Imaging Analyzer (BAS-2500; Fuji). The relative Rluc RNA level to mock control was plotted.Luciferase reporter assaysThe effect of PrV vhs on overall translation was evaluated by luciferase reporter assays. Firstly, 293T cells were seeded on the day before transfection at a density of 3-Fluoro-2-(trifluoromethyl)aniline 1 ? 105 cells/well in a 24-well plate. 800 ng of vhs plasmids were co-transfected with 800 ng of construct expressing luciferase by Lipofectamine 2000?reagent (Invitrogen) according to the manufacturer’s instructions. Renilla luciferase expression was detected at 24 h post-transfection using a Dual-Glo luciferase assay system (Promega) and measured by FLUOstar OPTIMA microplate reader (BMG Labtech, Offenburg, Germany).Statistical analysisRibonuclease activity of a thioredoxin-PrV vhs fusion protein has been previously described; nevertheless, the vhs assay used involved the degradation of RNA markers in reaction buffer at high concentrations of NaCl (0.3 M) and imidazole (0.25 M) [16]. Taking this into consideration, because the biochemistry properties of PrV vhsmediated enzyme activity still remains largely unknown, initially the nuclease activity of PrV vhs in the present study was measured following a well-established hydrolysis assay system [8, 18, 24]. To do.

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