| Trichinellosis is a world wide distributed zoonotic parasitic disease caused by nematode Trichinella spiralis which can infect more than 150 mammals including human. The disease could not only lead to enormously economic loss of meat industry but also severely threaten to the health of human. The complexity of Trichinella spiralis antigens and the difficulties to reproduce them in large quantity in vitro make it difficult for its immunodiagnosis and prevention. The newborn larvae (NBL) migrate to the striated muscles where they form a new type of syncitium known as the "nurse cell complex". The differences of NBL compared with adult worm and muscle larvae of Trichinella spiralis are not only in structures but also in components of antigens, all of which are strongly associated with the stage-specific expression of the nematode. The NBL is an important and invasive stage of Trichinella spiralis and it needs some particular nosogenetic factors to invade host. To investigate NBL stage expressed genes, which can help us well understand the molecular mechanism of NBL penetration and dedifferentiation during Trichinella spiralis infection. In previous study, we established a subtractive cDNA library of NBL by suppression subtractive hybridization (SSH), and a NBL stage-specific cDNA T314 was obtained. Using the T314 as a probe, the NBL cDNA library of Trichinella spiralis was screened, two different full length cDNAs of T314 (termed N5 and N10) were successfully found, they are in high homology and commonly encode DNase II. Thus, a DNase II gene family was suspected in the NBL cDNA library of Trichinella spiralis. We screened the NBL cDNA library of Trichinella spiralis at low hybridizing temperature using cDNA probe of N5. From 106 recombinant phages, 95 positive clones were obtained. The sequence analysis of them showed that there are 15 types of DNase II—13 novel cDNAs, 2 known cDNAs (N5 and N10). Database searching indicated that all of their encoded protein squences were homology to DNase II and formed a DNase II gene family. One of them, N10, was expressed and characterized. The stage-specific expressed gene of N10 contained a cDNA of 1233bp in full length with an open reading frame (ORF) of 1020bp. The ORF encoded a polypeptide of 340 amino acid residues with the predicted molecular weigh of 35.4KDa and isoeletric point of 9.53. Signal PV2.0 analysis showed that 1～18 amino acid residues of the peptide were signal peptide sequences. N10 deleted N-terminal signal peptide sequence by PCR was cloned into prokaryotic expression vector pET28a, and the recombinant vector pET-28a-N10 was successfully constructed. Then the recombinant plasmid was transformed into BL21star(DE3) and induced by IPTG.. The fusion protein of 38.7KDa was obtained from SDS-PAGE. The amount of expressed protein was increased with time extending and could reached 15% of the total bacterial protein quantity 4 hours after induction. The recombinant protein of N10 has an endonuclease activity that catalyzes the degradation of λDNA. Based on such kind of activity, the recombinant protein of N10 was predicted to have an endonuclease activity similar to DNase II. Furthmore, the DNase II-like activity of N10 measured by supercoiled plasmid DNA at pH5.0 was much higher than that observed under other conditions. However, the DNase II properties of Trichinella spiralis differ from those of other well-characterized molecules. Meanwhile, divalent cations had some effects on the DNase II-like activity. No endonuclease activity was detected in the same assays using control samples from empty-vector transfection, indicating that the DNase II-like activity of N10 was not due to contamination by endogenous vector.
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