Characterization and phylogenetic analysis of NDR domain-containing protein from Strongyloides ratti using Degenerate Primers-Based Polymerase Chain Reaction

The intestinal nematode Strongyloides ratti is important as an optimal model the human pathogen, S. stercoralis. A pair of degenerate primers was designed to amplify the target gene associated to the NDR domain-containing proteins based on the two conserved regions using the related nematode protein sequences. The putative NDR domain-containing gene fragment of 249 bp from the S. ratti was amplified using Touchdown RT-PCR. The results showed that the amplified fragment between these two motifs from S. ratti with the C-terminus part of reference protein has 66.3% similarities. By searching the EST (Expressed Sequence Tags) GenBank database, an overlapping 1082 bp cDNA fragment named SrNDR was compiled and found to contain a 972-nucleotide open reading frame that encodes a protein of 324 amino acids of 23.4 kDa expected molecular mass and a calculated pI of 7.64. The phylogram of SrNDR revealed that the gene variants of S. ratti SrNDR were grouped together with a strong bootstrap score of 87. Domain search analysis, showed the e-values of 1.05e-100 with the conserved NDR1 domain-containing of  hydrolase superfamily protein (pfam03096) between amino acid residues 9 to 287. The core domain of SrNDR folded with 12 alpha-helical conformation surrounded by 8 stranded antiparallel-beta-sheet. The three-dimensional structure model of SrNDR (residues 31-319) showed 100% similarity with human protein NDRG1 in 248 amino acids with 77% coverage. By using this program, we found a prediction of mostly a-helical (44.86%), Strand (1.08%) and random coils (54.05%) for the coding sequence of SrNDR. Based on the alignment of this protein with the corresponding nematodes, it can play a vital role in the survival of the parasite in the host cells.