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 Dbf۲-related (NDR) domain-containing proteins, based on two conserved regions identified from related nematode protein sequences. A putative NDR domain-containing gene fragment of ۲۴۹ 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 ۶۶.۳% similarities. By searching the EST (expressed sequence tags) GenBank database, an overlapping ۱۰۸۲ bp cDNA fragment named SrNDR was compiled and found to contain a ۹۷۲-nucleotide open reading frame encoding a protein of ۳۲۴ amino acids, with an expected molecular mass of ۲۳.۴ kDa and calculatal pI of ۷.۶۴. The phylogram of SrNDR revealed that the gene variants of S. ratti SrNDR were grouped together with a strong bootstrap score of ۸۷. Domain search analysis showed an e-value of ۱.۰۵e-۱۰۰ for the conserved NDR۱ domain within the a/b hydrolase superfamily protein (pfam۰۳۰۹۶), spanning amino acid residues ۹ to ۲۸۷. The core domain of SrNDR is folded into ۱۲ alpha-helices surrounded by eight antiparallel b-strands. The three-dimensional structure model of SrNDR (residues ۳۱-۳۱۹) showed ۱۰۰% identity to the human protein NDRG۱ across ۲۴۸ amino acids, with ۷۷% sequence coverage. Conclusion: By using this program, we found a prediction of predominantly a-helical (۴۴.۸۶%), Strand (۱.۰۸%) and random coil (۵۴.۰۵%) 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.

کلیدواژه ها:

Degenerate primers ، NDR domain-containing protein ، Touchdown reverse transcription polymerase chain reaction (RT-PCR)