Identification and characterisation of a cDNA sequence encoding a glutamic acid-rich protein specifically transcribed in Trichinella spiralis newborn larvae and recognised by infected swine serum
2002
Zarlenga, D.S. | Boyd, P. | Lichtenfels, J.R. | Hill, D. | Gamble, H.R.
Presently, little is known of the mechanism by which Trichinella penetrates and modulates reprogramming of muscle cells. In light of evidence demonstrating strong protective characteristics of antigens derived from this stage, understanding this process may shed light on potential targets for effective abatement of infection. To this end, a PCR-derived cDNA expression library was constructed using 0.5 micrograms of total RNA from Trichinella spiralis newborn larvae. The library consisted of >125,000 insert-containing clones. Approximately 40-50x10(3) clones were screened immunologically using sera from pigs experimentally infected with 7,000 Trichinella L1. Multiple clones reacting positively with the swine infection serum and encoding portions of a glutamic acid-rich protein were identified. Northern and Southern blots indicated at least two distinct genes that encoded the glutamic acid-rich proteins and that these genes were transcribed specifically in the newborn larvae stage. cDNA sequence data predicted open reading frames of 1,497 and 1,716 bp generating proteins of 498 amino acids and 571 amino acids, respectively. Both sequences consisted of approximately 39% glutamic acid and 16% serine residues, and differed by the presence of a 219 bp fragment present in the 1,716 bp sequence that was absent from the 1,497 bp sequence. PCR data indicated that additional isoforms exist within this gene family that are different in length from those described above. In addition, it was found that more than one isoform can exist within a single worm and that this pattern can vary between individual worms within a population. Mouse antibodies to recombinant antigen localised the glutamic acid-rich proteins to the periphery of the developing stichocyte cells within the newborn larvae consistent with the hypothesis that the newborn larval antigens are secreted.
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