Computational protocol: Prime-boost vaccination with recombinant protein and adenovirus-vector expressing Plasmodium vivax circumsporozoite protein (CSP) partially protects mice against Pb/Pv sporozoite challenge

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Protocol publication

[…] The recombinant proteins were characterised using a 12% sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) gel stained with Coomassie blue, immunoblotting, reverse-phase chromatography (RP-HPLC), and circular dichroism spectroscopy. Protein expression was confirmed by western blotting. For this analysis, the protein fractions were transferred from a 12% SDS-PAGE gel to a nitrocellulose membrane and exposed to mAbs [anti-His tag, 1:1,000, GE Healthcare; anti-PvCSP-VK210 (2F2), 1:1,000; and anti-PvCSP-VK247 (2E10.E9), 1:1,000 , followed by anti-mouse horseradish peroxidase (HRP)-conjugated IgG Ab (KPL). The bands were visualised using an ECL Western Blotting Analysis System (GE Healthcare).The high yield and purity of the recombinant proteins were confirmed by RP-HPLC using a Phenomenex Jupiter C18 column (4.6 mm × 250 mm, 5 µm particle, and 300 Å pore) with a Shimadzu Prominence HPLC System Solution (Shimadzu JPN Corp., Kyoto, KY). The HPLC procedure was performed using a 90% acetonitrile gradient in 0.1% trifluoroacetic acid at room temperature (RT, 22 °C) at a flow rate 1 mL/min for 30 min. A UV-visible absorbance detector (DAD: diode array detector, Shimadzu SPD M20A) was used for detection at 214 nm as described.The secondary structure of the recombinant proteins was analysed by circular dichroism (CD), using a JASCO-J815 spectropolarimeter (Jasco, Tokyo, Japan) at 25 °C. The purified proteins (diluted to 10 µM in phosphate buffered saline (PBS)) were loaded into a 5-mm quartz cuvette. Far-UV measurements (four scans) were obtained at wavelengths of 250–190 nm at 0.1-nm intervals, with a 1-nm bandwidth and 1 s response time. The spectrum presented is the average of the four scans, and the data obtained are reported as values of molar ellipticity [θ]MRW (deg × cm2 × dmol-1). A baseline measurement with PBS was subtracted from each protein spectrum. The data were smoothed with a Savitzky-Golay filter, and the percentages of secondary structure were predicted using the CD Analysis and Plotting Tool (http://capito.nmr.leibniz-fli.de/index.php). [...] All analyses were performed using GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA, USA) and graphics were plotted using SigmaPlot version 11.0 (GraphPad Software, La Jolla, CA, USA). A comparison of Ab titers in independent samples was performed by one-way analysis of variance (ANOVA). One-way ANOVA was also used to compare normally distributed log-transformed means for the different animal groups. Multiple comparisons were assessed by Tukey’s post-test with a significance level of P < 0.05. The significance in survival curves was determined using a log-rank Mantel-Cox test with a significance level of P < 0.05. Correlation coefficient analyses were determined with Pearson bivariate, two-tailed test of significance. […]

Pipeline specifications

Software tools CAPITO, SigmaPlot
Applications Miscellaneous, Circular dichroism spectroscopy
Organisms Plasmodium vivax, unidentified adenovirus, Plasmodium falciparum, Mus musculus, Komagataella pastoris, Homo sapiens, Pan troglodytes, Plasmodium berghei ANKA, Toxoplasma gondii