Computational protocol: Recombinant NAD-dependent SIR-2 Protein of Leishmania donovani: Immunobiochemical Characterization as a Potential Vaccine against Visceral Leishmaniasis

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Protocol publication

[…] Given seven sequences were aligned by using clustal W2 [, ] software at EBI. MSA file was generated in PHYLIP format and file so generated was then opened in Mega 5.2 tool [, ]. File was then converted to MEGA file format and using MEGA file format phylogenetic tree was built using MEGA 5.2 tool, using Maximum Parsimony approach []. Tool has been previous cited in other research articles [–] for constructing Phylogenetic tree. Given sequences were then uploaded in GLAM2 tool [] of MEME suite [] to identify motifs present in group of related protein sequences. This regular expression was queried in CDART tool [] to identify the functionality of motif. Same results were confirmed by Conserved Domain Database [] and SMART tool []. [...] Immune epitope database (IEDB) server ( was used for prediction of potential promiscuous T-cell epitopes on NAD sequence []. The IEDB uses information related to all experimentally determined immune epitope. It uses ANN, SMM, SMMPMBEC, CombLib Consensus, and NetMHCpan approach to predict the potential epitopes on user submitted protein sequence [–]. It covered wide range of MHC alleles. Both MHC-I and MHC-II based peptides were predicted. Server has been used for epitope prediction previously [–]. For prediction, we considered MHC alleles most prevalent in north Indian population []. [...] Glutaraldehyde cross-linking analysis with purified native SiR2 was carried out as described earlier []. To native SiR2 (100 μg/ml) was added an aliquot of 25% (v/v) glutaraldehyde (Sigma) to achieve a final concentration of 1% (v/v) glutaraldehyde and incubated at 25°C for 5min. Cross linking sample was quenched by addition of 200mM sodium borohydride. After 20min incubation, 3μl of 10% (w/v) aqueous sodium deoxycholate was added. The pH of the reaction mixture was decreased to 2–2.5 by the addition of orthophosphoric acid that resulted in precipitation of the cross-linked SiR2 protein. After centrifugation (13,000×g, 4°C, 10 min) the obtained precipitates were re-dissolved in 0.1M Tris—HCl (pH 8.0), 1% SDS (w/v) and 50mM DTT and heated at 90–100°C. The molecular mass of the crosslinked products were determined by 6% (w/v) SDS—PAGE. Enzyme activity for SIR2 deacetylase was determined fluromatric using CycLex SIR2 deacetylase assay kit with some modifications []. Briefly enzyme reaction was performed in a 96 well plate consisting fluorescence-labelled acetylated peptide as substrate for deacetylase, NAD and lysylendopeptidase in buffer (Tris-HCl, pH 8.8). Reaction was initiated by adding purified recombinant SIR2 deacetylase protein. Fluorescence was measured with excitation at 490 nm and emission at 530 nm. [...] QRT-PCR was performed to assess the expression of mRNAs for various cytokines and iNOS in splenic cells. Splenic tissues were taken from each of the three randomly chosen animals. Total RNA was isolated using Tri-reagent (Sigma-Aldrich) and quantified by using Gene-quant (Bio-Rad). One microgram of total RNA was used for the synthesis of cDNA using a first-strand cDNA synthesis kit (Fermentas). For real-time PCR, primers were designed using Beacon Designer software (Bio-Rad) on the basis of cytokines and iNOS mRNA sequences available on PubMed (). qRT-PCR was conducted as per the protocol described earlier [] by using 12.5 μl of SYBR green PCR master mix (Bio-Rad), 1 μg of cDNA, and primers at a final concentration of 300 nM in a final volume of 25 μl. PCR was conducted under the following conditions: initial denaturation at 95°C for 2 min followed by 40 cycles, each consisting of denaturation at 95°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 40 s per cycle using the iQ5 multicolor real-time PCR system (Bio-Rad). cDNAs from normal hamsters were used as “comparator samples” for quantification of those corresponding to test samples whereas in vaccination studies, cDNAs from infected hamsters were used as “comparator samples”. All quantifications were normalized to the housekeeping gene HPRT. A no-template control c-DNA was included to eliminate contaminations or nonspecific reactions. The cycle threshold (CT) value was defined as the number of PCR cycles required for the fluorescence signal to exceed the detection threshold value (background noise). Differences in gene expression were calculated by the comparative CT method []. This method compares test samples to a comparator sample and uses results obtained with a uniformly expressed control gene (HPRT) to correct for differences in the amounts of RNA present in the two samples being compared to generate a ΔCT value. Results are expressed as the degrees of difference between ΔCT values of test and comparator samples. The level of IgG antibody and its isotypes in sera samples of hamsters of different experimental groups was measured as per protocol by Samant et al. [] with slight modifications. Briefly, 96-well ELISA plates (corning) were coated with rLdSir2RP (0.2 μg/100 μl/well) overnight at 4°C and blocked with 1.5% BSA at room temperature for 1 h. Sera was used at a dilution of 1/100 for IgG, IgG1, and IgG2 and kept for 2h at RT. Biotin-conjugated mouse anti-Armenian and Syrian hamster IgG, IgG1 and biotinylated anti-Syrian hamster IgG2 (BD Pharmingen) were added for 1h at room temperature at 1/1000 dilutions and were further incubated with peroxidase-conjugated streptavidin at 1/1000 (BD Pharmingen) for 1 h. Finally, the substrate O-phenylenediamine dihydrochloride (Sigma) was added and the plate was read at 492 nm. […]

Pipeline specifications

Software tools cycleX, Beacon Designer
Applications scRNA-seq analysis, qPCR
Organisms Leishmania donovani, Homo sapiens, Mesocricetus auratus
Diseases Leishmaniasis, Leishmaniasis, Visceral, Protein Deficiency
Chemicals NAD