Characterization and Phylogenetic Analysis of NDR Domain Protein From Strongyloides ratti Using Degenerate Primer PCR

Introduction: The intestinal nematode Strongyloides ratti is an important and optimal model for the human pathogen, Strongyloides stercoralis. To design degenerate primers and amplify a putative NDR domain‑containing gene fragment from Strongyloides ratti using touchdown reverse transcription polymerase chain reaction (RT-PCR).
Materials & Methods: A pair of degenerate primers was designed to amplify the target gene associated with nuclear Dbf2-related (NDR) domain-containing proteins, based on two conserved regions identified from related nematode protein sequences. A putative NDR domain-containing gene fragment of 249 bp from the S. ratti was amplified using Touchdown RT-PCR. 
Results: The results showed that the amplified fragment between these two motifs from S. ratti, aligned with the C-terminus of the reference protein, shares 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 encoding a protein of 324 amino acids, with an expected molecular mass of 23.4 kDa and calculatal 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 an e-value of 1.05e-100 for the conserved NDR1 domain within the a/b hydrolase superfamily protein (pfam03096), spanning amino acid residues 9 to 287. The core domain of SrNDR is folded into 12 alpha-helices surrounded by eight antiparallel b-strands. The three-dimensional structure model of SrNDR (residues 31-319) showed 100% identity to the human protein NDRG1 across 248 amino acids, with 77% sequence coverage. 
Conclusion: By using this program, we found a prediction of predominantly a-helical (44.86%), Strand (1.08%) and random coil (54.05%) content for the coding sequence of SrNDR. Based on the alignment of this protein with homologous sequences from related nematodes, it may play a vital role in parasite survival within host cells.