[Cloning, expression and function analysis of thioredoxin-1 protein of Schistosoma japonicum].
摘要
OBJECTIVE: To clone and express recombinant thioredoxin-1 protein of Schistosoma japonicum (SjTrx-1), determine its enzyme activity, analyze the stage-specific transcription of SjTrx-1 gene and observe the distribution of SjTrx-1 protein in adult worm. METHODS: SjTrx-1 coding sequence identified from S. japonicum cDNA library was amplified by PCR. Subsequently, the obtained target fragment was subcloned into pET28a, and the recombinant plasmid was then transformed into E. coli BL21 (DE3) cells and induced with IPTG for protein expression. The recombinant SjTrx-1 protein was purified with Ni-NTA resin and the enzyme activity was determined by DTT/insulin reduction method. The rabbit antiserum was prepared by immunizing New Zealand white rabbit with purified recombinant SjTrx-1 protein. Indirect immunofluorescence staining with the prepared polyclonal antibody was used to investigate the distribution of SjTrx-1 protein in adult worm. Total RNA was extracted from different stages of S. japonicum and reverse-transcribed into cDNA samples. RT-PCR was used to analyze the SjTrx-1 transcription level of each development stages. RESULTS: The expression vector of SjTrx-1/pET28a was established and the recombinant SjTrx-1 protein was expressed as soluble form in E. coli (about M(r) 38 000). The purified Sjrx-1 protein was obtained through Ni-NTA affinity purification and was demonstrated with adequate enzyme activity in vitro. The stage-specific analysis showed that SjTrx-1 was expressed in each of the developmental stages studied, with increased level in sporocysts, schistosomula and adult worms, and relatively low level in miracidia and cercaria. Immunolocalization revealed SjTrx-1 was widely spread in the worm without tissue-specific. CONCLUSION: SjTrx-1 is an abundant and widely distributed protein with increased level in sporocysts, schistosomula and adult worms.