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8 results for “Baculovirus expression system”
Fig. 5 in Successful transcription but not translation or assembly of Solenopsis invicta virus 3 in a baculovirus-driven expression system
Fig. 5. Confirmation of heterologous expression of SINV-3 transcript by amplification of the 3' (A) and 5' termini (B) from RNA templates purified from SINV- 3-transfected Sf21 cells. Regions amplified are illustrated in the genome diagram between (A) and (B). (A) Three plaque preparations (AcSINV-3 CiC, AcSINV-3 CiD, and AcSINV-3 DiD) were separated by centrifugation into soluble and pelleted fractions, treated with DNase I, reverse transcribed, and the 3' end of the genome amplified by PCR. Lane assignments were as follows: 1 = mass marker (bp); 2, 6 = AcSINV-3 CiC; 3, 7 = AcSINV-3 CiD; 4, 8 = AcSINV-3 DiD; 5, 9 = mock infection; 10 = positive control (wild-type virus); 11 = negative control; 12 = non-template control. (B) AcSINV-3 plaque preparations (CiC and DiD) evaluated by PCR of the 5' end of the genome (RNA preparations). Lane assignments were as follows: 1 = mass marker (bp); 2, 3 = DNase treated, reverse transcribed; 4, 5 = without DNase treatment, reverse transcribed; 6, 7 = DNase treated, without reverse transcription; 8, 9 = without DNase treatment, without reverse transcription; 10 = positive control; 11 = negative control; 12 = non-template control. (C) PCR amplification of the entire SINV-3 genome from DNA preparations of AcSINV-3 CiC (lane 2) and AcSINV-3 DiD (lane 3). Lane 1, molecular markers; lane 4, non-template control. (D) Western blot to evaluate translation of the SINV-3 transcript by detection of viral capsid protein 2 (VP2). Lane assignments were as follows: 1 = AcSINV-3 CiC (4 dpi); 2 = AcSINV-3 CiC (5 dpi); 3 = AcSINV-3 CiD (4 dpi); 4 = AcSINV-3 DiD (5 dpi); 5 = mock infection (negative control); 6 = positive control (purified wild-type SINV-3; kDa).
Fig. 3 in Successful transcription but not translation or assembly of Solenopsis invicta virus 3 in a baculovirus-driven expression system
Fig. 3. (A) Plaque assay results for recombinant SINV-3 (AcSINV-3) transfection of Sf21 cells indicating the dilution used for each plate. (B upper panel) Representa- tive plaque with Sf21 cells stained with neutral red 10 d afer transfection (magnified 100 times). (B lower panel) Corresponding mock-infected Sf21 cells (negative control) afer 10 d of exposure. Plaque areas identify infection of insect cells by virus with corresponding cell death.
Fig. 2 in Successful transcription but not translation or assembly of Solenopsis invicta virus 3 in a baculovirus-driven expression system
Fig. 2. (A) pFastBac1_SINV-3 hybrid construct map. Locations of the restriction sites (black hash marks), bacterial transposon Tn7 sites (grey triangles), polyhedrin promoter (angled arrow corresponding to the sequence below), SINV-3 open reading frames (dark closed arrows), and approximate location of the area detected by the polyclonal antibody preparation (pAb) are shown. (B) Verified sequence of the pFastBac1_SINV-3 hybrid construct illustrating the late gene polyhedrin promoter (angled arrow). SINV-3 sequence is in bold font,with pFastBac1 sequence in normal font, and restriction sites are superscripted and corresponding sequences italicized.Underlined sequence represents the inserted late gene polyhedrin core promoter.Analyzed sequences of the 5'and 3'termini and restriction sites were identical to the wild-type virus.
Fig. 1 in Successful transcription but not translation or assembly of Solenopsis invicta virus 3 in a baculovirus-driven expression system
Fig. 1. Schematic of the SINV-3 genome and sub-cloning strategy to assemble the pFastBac1 donor plasmid/SINV-3 construct. (A) Organization of the SINV-3 genome illustrating the 2 ORFs numbered 1 and 2 that encode for non-structural and structural proteins, respectively, and the genome sections sub-cloned. Oligonucleotide primers used to generate cDNA and amplify each section are indicated. Primers with introduced restriction sites are also indicated. (B) Unique restriction sites for each sub-clone and the assembly process employed to concatenate the entire SINV-3 genome in the pFastBac1 donor vector.
Large scale expression and purification of full length huntingtin Q46 from baculovirus expression system (2016/09/04)
<p>Open lab notebook huntingtin structure function project.<br> </p>
Test expression and purification of full length huntingtin Q23, Q46 and Q78 in baculovirus expression system (2016/08/04)
<p>Open lab notebook huntingtin structure function project.</p>
Large scale expression and purification of full length huntingtin Q23 from baculovirus expression system (2016/08/29)
<p>Open lab notebook huntingtin structure function project.<br> </p>
Fig. 4 in Successful transcription but not translation or assembly of Solenopsis invicta virus 3 in a baculovirus-driven expression system
Fig. 4. Quantitative PCR (absolute) results evaluating transcript production of SINV-3 by AcSINV-3-infected Sf21 cells. RNA preparations from AcSINV-3 (2 rep- licates, Ci and Di) were treated with DNase I, reverse transcribed, and amplified by qPCR. Results were compared with a series of plasmid constructs containing a portion of the SINV-3 genome (102–109 genome equivalents). The region amplified was at the 3'-most end of ORF2 (see Fig. 2). No amplification was detected in polyhedrin-negative AcRP23.lacZ preparations.
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OpenNeuro
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