Identification, isolation, and cloning of the full-length sequence of a gene encoding trypsin inhibitor-like protein (TIL) secreted by the intestinal parasitic nematode Strongyloides ratti

Abstract

BackgroundIntestinal nematodes may interact with the intestinal mucosal host cells through molecules they produced actively in the host environment. Many excretory/secretory products (ESP) of the intestinal nematodes are considered as interesting candidates to be examined in vitro on their tolerance-inducing activities. Nematode-derived protease inhibitors are believed to have multiple, specific abilities for the host immunomodulation, playing on various immune effector mechanisms. Among the previously identified 500 ESP from Strongyloides ratti, trypsin inhibitor-like (Sra-TIL) peptides were detected in high concentration in the ESP of the parasitic female (pF). The aim is to identify interesting tolerance-inducing proteins belonging to the longtime adapted human biome which may be potentially applied to moderate inflammatory immune reactions.ResultsIn the present study, the full-length cDNA (from the parasitic female) and gDNA of the ~ 1605 bp Sra-til is identified and isolated by polymerase chain reaction (PCR), subsequent to cluster/EST analysis via alignment and BLAST search. gDNA and RNA were extracted from infective third stage larvae (iL3) and pF respectively, and the cDNA was synthesized.Then, the Sra-til was cloned and sequenced. The genomic organization analysis revealed that introns were absent in the Sra-til gene structure. Sequence analyses of both nucleotide and predicted protein sequences were investigated using different bioinformatic tools. The predicted architecture of the translated amino acid sequence showed the presence of a signal peptide for secretion. The architecture of Sra-TIL was Sra-TIL = TIL, EGF (epidermal growth factor) × 6). Phylogenetic analysis of the deduced amino acid sequence showed how related this novel Strongyloides protein to TIL orthologs from other nematodes.ConclusionThe full-length Sra-til gene which encodes an interesting ESP candidate, secreted from pF, was identified, isolated, cloned, and then sequenced. Both nucleotide and amino acid sequences were analyzed by various bioinformatic tools. The predicted innovative structural advantages of Sra-TIL polypeptides secreted from a parasitic intestinal stage of S. ratti nematode is probably reflective of the abundance of different biological functions, including the protection against host proteases, host immunomodulation via proliferation induction of the damaged intestinal cells as well as wound healing properties.