Cloning and expression of Pagumogonimus skrjabini cysteine protease cDNA

Aim To clone and express the cysteine protease cDNA fragment of Pagumogonimus skrjabini metacercarias.Methods The cysteine proteinase cDNA fragment was amplified by RT-PCR with degenerated primers.The production was TA-cloned into the pUCm-T vector and sequenced.DNASIS program was used to analyse the nucleotide sequence and deduce the amino acide sequence,which was aligned with the correlated parasite cysteine protease afterwards.After the ligation of PinPoint TM Xa-1 T-vector and PCR product,the clones were screened to determine the fragment orientation prior to protein expression.The expression was carried out in E.coli and analyzed on SDS-PAGE.The expressed fusion protein was identified and its immunoreactive ability was determined by Western blotting.Results A 498bp cDNA fragment was amplified by RT-PCR and sequenced.An amino acide sequence of 166 was deduced by DNASIS.Sequence analysis and alignment showed significant homologies with the correlated parasite cysteine proteases and conservation of Cys,His and Asn residues that form a catalytic triad.In the identification and immunoreaction observation of the expressed fusion protein,Western blot illustrated a band of 33kDa in cells containing the PinPoint TM Xa-1 T-Vector plus the insert of interest.Conclusion The cysteine protease cDNA fragment from Pagumogonimus skrjabini metacercaria was cloned.And a fusion protein with 33kDa molecular weight is expressed.The expressed protein has immunoreactive abilitys as it reacted with immune serum.