The study for gene cloning and recombinant protein expression of endoribonuclease of E. cinetorchis

초록

The adult worm of Echinostoma cinetorchis, oriental intestine fluke, is known to migrate to the intestine in mammalian hosts and cause significant pathological changes in the small intestine. The aim of this study is to determine the full length DNA sequence of endoribonuclease of Echinostoma cinetorchis and elucidate its molecular characteristics. Adult worms of Echinostoma cinetorchis were obtained from sprague-Dawley rats infected orally with metacercariae which were isolated from naturally infected snale (Segmentina hemisphaerula). Endoribonuclease of Echinostoma cinetorchis consists of 927 nucleotides which encoded 308 amino acids. Predicted molecular weight of it was 34 kDa. Sequence analysis and alignment showed low similar to the other members of the parasites as under 40% value, predicted as a novel gene. The cloned gene of endoribonuclease was expressed in Escherichia coli and purified with GST column. The activity of purified recombinant endoribonuclease was verified by designed enzyme activity assay, which cleaves the 3’-O-P bond of RNA in a DNA-RNA duplex for producing 3’-hydroxyl and 5’-phosphate. The localization of endoribonuclease were investigated by immunohistochemistry (IHC) assay for verifying its locations in the muscle layer of Echinostoma cinetorchis. These gene provide molecular probes for further analysis of the structure and function of these important metabolic enzymes.