Molecular cloning and characterization of transgelin-like proteins mainly transcribed in newborn larvae of Trichinella spp.

A cDNA library was constructed from Trichinella pseudospiralis muscle larvae. One cDNA clone, designated Tp4, contained a cDNA transcript of 783 bp in length, with a single open reading frame that encoded 153 amino acids (16,793 Da as the estimated molecular mass). The predicted amino acid sequence of Tp4 showed that the clone had a calponin homology domain and was approximately 50% identical to the transgelin-like proteins (calponin-family members) present in Bombyx mori or Tribolium castaneum. A homologue of the Tp4 clone was also present in cDNA from Trichinella spiralis, and this clone was designated Ts4. A comparison of the amino acid sequence of the transgelin-like proteins from T. spiralis (Ts4 protein) with the Tp4 protein indicated that the two proteins are very similar (about 94% homology). Real time quantitative polymerase chain reaction results showed that the transcription level of the Tp4 and Ts4 genes was highest in newborn larvae. On Western blot, the recombinant Tp4 and Ts4 proteins migrated at 20 kDa when reacted to an antibody against the recombinant Tp4 and Ts4 proteins, respectively. An antibody against the recombinant Tp4 and Ts4 proteins strongly stained two bands migrating at approximately 9 and 8 kDa in the crude extracts from adult worms and newborn larvae, but only weakly stained proteins in muscle larvae. However, an immunocytochemical study showed that the Tp4 protein was present within the muscle of the muscle larvae of T. pseudospiralis. The antibody level against the recombinant Tp4 antigens in infected mice began to increase from 8 days post-infection, was highest in 13 days post-infection, and then slowly decreased.