Cloning and analysis of a novel cDNA from Trichinella spiralis encoding a protein with an FYVE zinc finger domain

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Abstract

A cDNA library from Trichinella spiralis adults 3 days post-infection was screened with a cDNA probe, designated T54, derived from a newborn larvae subtracted cDNA library. Sequence analysis showed that the positive clone contained a cDNA insert of 1464 bp in length with a single open reading frame of 1290 bp, which encoded a protein of 429 amino acids with a putative molecular mass of 49.9 kDa. Database analysis predicted the deduced protein had a leucine zipper motif and an FYVE zinc finger domain. The recombinant fusion protein was expressed and rabbit anti-recombinant protein sera reacted with a single peptide migrating at approximately 55 kDa in crude worm extract from muscle larvae, adults and newborn larvae stages.

Introduction

Trichinellosis is a parasitic disease with a worldwide distribution caused by Trichinella spp. It remains a serious public threat in both developed and developing countries (Murrel and Pozio, 2000, Liu and Boireau, 2002). Trichinella antigens have shown stage specificity, changing qualitatively and quantitatively in different development stages (Parkhouse and Ortega-Pierres, 1984, Boireau et al., 1997). Cloning and identification of differentially expressed genes will help to understand not only the function in development and differentiation but also the molecular mechanisms of parasite and host interaction. To this end, a newborn larvae (NBL) subtracted cDNA library was constructed to enrich NBL stage specific genes using suppression subtractive hybridization (Diatchenko et al., 1996). Sequence analysis of the NBL subtracted cDNA clones showed that one clone, designated pT-Adv-T54, contained a cDNA insert which encoded a putative polypeptide sharing 43% similarity with an adult-specific antigenic protein of Angiostrongylus cantonensis (Bessarab and Joshua, 1997). Herein, we clone and characterize the full-length cDNA.

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Parasites and cDNA library construction

Trichinella spiralis (ISS534) muscle larvae (ML) were recovered from mice 35 days post-infection by artificial digestion. Adult worms were obtained from rat intestine of 3 days post-infection (Ad3) and 5 days post-infection (Ad5). Newborn larvae were recovered by incubating 6-day-old adults in RPMI 1640 medium for 24 h followed by sieving and centrifugation. Stage specific NBL subtracted cDNA library was constructed by subtracting ML/Ad cDNA from NBL cDNA. Suppression subtractive hybridization

Screening of cDNA library and sequence analysis

Sequence analysis of clone pT-Adv T54 revealed a cDNA fragment encoding a peptide with 43% identity to residues 214–414 of a Angiostrongylus cantonensis protein, which is a 66 kDa, adult-specific and antigenic protein (Bessarab and Joshua, 1997). After tertiary screening from 5 × 104 recombinant phages of Ad3 cDNA library, four positive clones were sequenced on both strands. The resultant consensus cDNA sequence was 1464 bp in length with an open reading frame (ORF) of 1290 bp. The deduced

Discussion

The leucine zipper is present in many gene regulatory proteins and DNA binding proteins (Landschulz et al., 1988). The FYVE finger is a cysteine-rich zinc-finger-like motif that coordinates two zinc atoms (Stenmark and Aasland, 1999) and the function of the FYVE domain is to target signal-transducing proteins to cell membranes through binding to the membrane lipid phosphatidylinositol-3-phosphate with high specificity (Gaullier et al., 1998). The overall architecture and size of this protein

Acknowledgements

This work was supported by grants of EU TRICHIPORSE QLRT-2000-01156, PRA BT 03-02 and NSFC 30170709, 30328020 (National Natural Science Foundation of China).

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The nucleotide sequence data reported in this paper are available in the GenBank database under the accession number AY125954.

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