Computational protocol: Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

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Protocol publication

[…] RT1.14-expressing HeLa were lysed and analyzed by Western blotting using polyclonal rabbit anti-RT [] or monoclonal murine anti-RT antibodies [] as primary and anti-rabbit or anti-mouse HRP-conjugated antibodies as secondary (Jackson, USA) as was previously described []. Cell culture fluids were centrifuged at 5000 ×g and then concentrated approximately 10 times with Vivaspin 500 units (Sartorius Stedim, Germany) with a 30,000 MWCO membrane. Concentrated culture fluid was mixed with Laemmli buffer and then subjected to Western blotting same way as the cell lysates. Immune complexes on the membrane were detected with ECL (Amersham, USA) and X-ray film (FujiFilm, Japan). The data was processed in ImageJ software (http://rsb.info.nih.gov/ij). After RT-specific staining, blots were washed and restained first with monoclonal anti-β-actin murine antibodies (Sigma, USA) and then with anti-mouse HRP-conjugated antibodies (Dako, Denmark). To assess the level of RT expression per cell, the percent of cells expressing RT was estimated from the efficacy of transfection established in a control cotransfection with GFP plasmid (peGFP-N1, Novagen, Germany) used as a reporter. The number of cells for each sample was counted with hemocytometer and certain number of cells was taken for Western blotting analysis. The number of transfected cells was estimated from the percentage of transfection efficacy. The amount of RT protein in the lysed cells and culture fluids was calculated based on a standard curve built using the recombinant RT 1.14 protein and dispensed in serial dilutions in the concentration range from 1 to 20 ng per well; the latter samples were analyzed together with the lysates as described earlier []. RT content per cell was calculated by dividing these values by the number of transfected cells. [...] Statistical evaluations were done using STATISTICA AXA 10.0 (StatSoft Inc., OK, USA). Nonparametric statistics was chosen as appropriate for sample sizes <100 entries. Continuous but not normally distributed variables, such as the average radiance in photons/s/cm2/Sr, antibody titers, or the number of cytokine-producing SFCs, cytokine levels in pg/ml, were compared in groups by the nonparametric Kruskal-Wallis and pairwise by Mann–Whitney U test. Correlations were run using the Spearman rank order test. p values <0.05 were considered significant. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Human immunodeficiency virus 1, Mus musculus, Tick-borne encephalitis virus
Diseases HIV Infections