Immunoreactivity and Molecular Dynamics of arginine kinase wild type from tick Rhipicephalus sanguineus and a mutant E1
The brown dog tick Rhipicephalus sanguineus is a significant vector for Rocky Mountain Spotted Fever (RMSF), a disease caused by Rickettsia rickettsii. We report a data on the effect of the tick bite on the immune response in humans of arginine kinase wild type (WT) from tick Rhipicephalus sanguineus and a mutant E1. Absorbance values obtained by ELISA, Calculation of INDEX Value and statistical analysis data is showed. Furthermore, predictions of WT and E1 RsAK structures were conducted using ColabFold v1.5.2 software. The impact of the mutation on conformational stability was evaluated through molecular dynamics (MD) simulations.
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We used thirty sera samples from patients diagnosed with Rocky Mountain Spotted Fever (RMSF) disease by immunofluorescence with a 1:128 titer for the immunoreactivity tests. Those samples were obtained from the Laboratorio Estatal de Salud Pública de Sonora. Also, ten samples from healthy persons from a local blood bank were secured to be used as controls. The wells of a 96-well polystyrene microplate were coated with either 50 µL of 0.3 µM of WT RsAK or the E1 mutant in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), and incubated at 4 °C overnight. The wells were then blocked with TBST (Tris HCl 0.04 M, NaCl 0.138 M, KCl - 0.0027 M, Tween 20 - 0.05%, pH 8.0) using 0.1% blotting grade blocker and 1% bovine serum albumin for 1 hour, followed by a wash. The sera of thirty RMSF patients and ten blood bank donors were tested against the WT and E1 RsAK. All sera were diluted 1:100, and 50 µL was applied to each well. After a 1-hour incubation, the microplates were washed, and 50 µL of 1:4000 diluted anti-human IgG conjugated with alkaline phosphatase were added, incubated for 30 min, and washed. The color was developed for 30 min with an alkaline phosphatase substrate from Bio-Rad®, and the plate was read at 415 nm. Absorbance data were further analyzed in terms of the Index value. To establish a threshold value, the average absorbance of the controls was calculated, and then two standard deviations were added individually for each protein. The Index value was obtained by dividing each sample data by the threshold value. Samples with an Index value of 1 or higher were considered positive for each antigen. The data were subjected to a two-tailed paired Student's t-test (p<0.05), followed by a one-tailed paired Student's t-test to assess significant differences in interaction magnitude among the antibodies to WT RsAK, E1, and between the RMSF and control sera.
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